Combination vaccines with serogroup b meningococcus and d/t/p

ABSTRACT

Serogroup B meningococcus antigens can successfully be combined with diphtheria, tetanus and pertussis toxoids (“DTP”) to provide effective combination vaccines for protecting against multiple pathogens. These combinations are effective with a range of different adjuvants, and with both pediatric-type and booster-type DTP ratios. The adjuvant can improve the immune response which the composition elicits; alternatively, by including an adjuvant it is possible for the compositions to have a relatively lower amount of antigen while nevertheless having immunogenicity which is comparable to unadjuvanted combination vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/420,238, with an international filing date of Sep. 6, 2013, now U.S.Pat. No. 9,526,776; which is the National Phase of PCT Application No.PCT/EP2013/068414, filed Sep. 6, 2013; which claims the benefit of U.S.Provisional Application No. 61/697,756, filed Sep. 6, 2012; all of whichare incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 303822012710SeqList.txt,date recorded: Dec. 16, 2016, size: 122 KB).

TECHNICAL FIELD

This invention is in the field of combination vaccines i.e. vaccinescontaining mixed immunogens from more than one pathogen, such thatadministration of the vaccine can simultaneously immunize a subjectagainst more than one pathogen.

BACKGROUND ART

Vaccines containing antigens from more than one pathogenic organismwithin a single dose are known as “multivalent” or “combination”vaccines. Various combination vaccines have been approved for human use,including trivalent vaccines for protecting against diphtheria, tetanusand pertussis or against measles, mumps and rubella. These vaccinesoffer patients the advantage of receiving a reduced number ofinjections, which can lead to the clinical advantage of increasedcompliance (e.g. see chapter 29 of ref 1), particularly in pediatricpatients.

One difficulty when providing new combination vaccines is the potentialfor adverse vaccine-vaccine interactions between the mixed components,which may be due to physical or chemical factors. For instance,reference 2 discusses potential alterations in immunogenicity whenantigens are combined, and reference 3 reports that the development ofcombination vaccines involves much more than the simple mixing ofexisting antigens. Similarly, reference 4 reviews a variety ofclinically-relevant interactions (see also reference 5), and reference 6reviews the technical challenges faced when making a combinationvaccine.

It is an object of the invention to provide further and improvedcombination vaccines, and in particular those which can protect againstserogroup B meningococcus and other pathogens.

SUMMARY OF THE INVENTION

The inventors have shown that serogroup B meningococcus antigens cansuccessfully be combined with diphtheria, tetanus and pertussis toxoids(“DTP”) to provide effective combination vaccines for protecting againstmultiple pathogens. These combinations are effective with a range ofdifferent adjuvants, and with both pediatric-type and booster-type DTPratios. The adjuvant can improve the immune response which thecomposition elicits; alternatively, by including an adjuvant it ispossible for the compositions to have a relatively lower amount ofantigen while nevertheless having immunogenicity which is comparable tounadjuvanted combination vaccines.

In general, therefore, the invention provides an immunogenic compositioncomprising (a) a serogroup B meningococcus immunogen and (b) at leastone of a diphtheria toxoid, a tetanus toxoid, and/or a pertussis toxoid.The composition will usually also include an adjuvant, such as analuminium salt or an oil-in-water emulsion. Preferably component (b)includes all three of a diphtheria toxoid, a tetanus toxoid, and apertussis toxoid. In some embodiments component (b) includes morediphtheria toxoid than tetanus toxoid (measured in Lf units), but inother embodiments it includes more tetanus toxoid than diphtheriatoxoid.

In a first embodiment the invention provides an immunogenic compositioncomprising: (a) a serogroup B meningococcus immunogen; (b) a diphtheriatoxoid, a tetanus toxoid, and a pertussis toxoid; and (c) an adjuvant.The adjuvant can comprise one or more of an aluminium salt adjuvant, aTLR agonist, or an oil-in-water emulsion.

In a second embodiment the invention provides an immunogenic compositioncomprising: (a) a serogroup B meningococcus immunogen; and (b) adiphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, wherein thediphtheria toxoid is present in an excess relative to tetanus toxoid asmeasured in Lf units. This composition can also include an adjuvant, andthis can comprise one or more of an aluminium salt adjuvant, a TLRagonist, or an oil-in-water emulsion.

In a third embodiment the invention provides an immunogenic compositioncomprising: (a) a serogroup B meningococcus immunogen; and (b) adiphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, wherein thetetanus toxoid is present in an excess relative to diphtheria toxoid asmeasured in Lf units. This composition can also include an adjuvant, andthis can comprise one or more of an aluminium salt adjuvant, a TLRagonist, or an oil-in-water emulsion.

Compositions of the invention can include antigens in addition todiphtheria toxoid, tetanus toxoid, and pertussis toxoid e.g. they caninclude Hib capsular saccharide (ideally conjugated), HBsAg, IPV,meningococcal capsular saccharide (ideally conjugated), etc.

Serogroup B Meningococcus Immunogens

Immunogenic compositions of the invention include a serogroup Bmeningococcus immunogen. When administered to human beings (or to asuitable animal model) the immunogen can elicit a bactericidal immuneresponse. These immunogens can be proteins, liposaccharides, orvesicles.

Various serogroup B meningococcus protein immunogens are known in theart, including but not limited to NHBA, fHbp and NadA as found in theBEXSERO™ product [7,8]. Further protein immunogens which can be includedin compositions of the invention are HmbR, NspA, NhhA, App, Omp85, TbpA,TbpB, Cu,Zn-superoxide dismutase, and ZnuD. Further details of theseimmunogens are discussed below.

A vaccine may include one or more of these various immunogens e.g. itcan include each of NHBA, fHbp and NadA. It can also include variantforms of a single immunogen e.g. it can include more than one variant ofmeningococcal fHbp (i.e. two fHbp proteins with different sequences[191, 9]).

The serogroup B meningococcus protein immunogens can be present asfusion proteins. For instance, the BEXSERO™ product includes two fusionproteins: SEQ ID NO: 4 is a fusion of NMB2091 and a fHbp; and SEQ ID NO:5 is a fusion of a NHBA and NMB1030. One useful fusion protein is SEQ IDNO: 19, which includes NMB2091 and two copies of a fHbp.

Two useful combinations of serogroup B immunogens include: a NHBA e.g.SEQ ID NO: 5; a fHbp e.g. either SEQ ID NO: 4 or SEQ ID NO: 19; and aNadA e.g. SEQ ID NO: 6. Other useful combinations include proteins whichdiffer from SEQ ID NOs: 5, 4, 19 & 6 by up to 5 amino acids each butwhich retain the ability to elicit antibodies which recognise SEQ IDNOs: 5, 4, 19 & 6.

Compositions which include at least one fHbp immunogen are preferrede.g. those containing two different fHbp sequences. Details of suitablefHbp combinations are discussed below.

Thus compositions of the invention can usefully include (a) the mixtureof three serogroup B meningococcus protein immunogens disclosed as‘5CVMB’ in reference 8 or (b) the mixture of serogroup B meningococcusprotein immunogens disclosed as ‘rLP2086’ in reference 10.

Usually, the serogroup B meningococcus immunogens are purified solublerecombinant proteins. In some embodiments, however, they can be presentin meningococcal vesicles. Thus the composition can includemcningococcal vesicles i.e. any protcoliposomic vesicle obtained bydisruption of or blebbing from a meningococcal outer membrane to formvesicles therefrom that retain antigens from the outer membrane. Thusthis term includes, for instance, OMVs (sometimes referred to as‘blebs’), microvesicles (MVs) and ‘native OMVs’ (‘NOMVs’). Various suchvesicles are known in the art (e.g. see references 11 to 25) and any ofthese can be included within a composition of the invention. Furtherdetails of these vesicles are given below. In some embodiments, however,the composition is vesicle-free.

A composition of the invention can preferably elicit a serumbactericidal assay after being administered. These responses areconveniently measured in mice and are a standard indicator of vaccineefficacy. Serum bactericidal activity (SBA) measures bacterial killingmediated by complement, and can be assayed using human or baby rabbitcomplement. For instance, a composition may induce at least a 4-foldrise in SBA in more than 90% of recipients.

A composition of the invention can preferably elicit an immune responsein human beings which is protective against serogroup B meningococcus.For instance, the vaccine may elicit an immune response which isprotective at least against a prototype serogroup B strain such as MC58,which is widely available (e.g. ATCC BAA-335) and was the strainsequenced in reference 26. Other strains can also be used, but aresponse against MC58 is easily tested.

Diphtheria Toxoid

Diphtheria is caused by Corynebacterium diphtheriae, a Gram-positivenon-sporing aerobic bacterium. This organism expresses aprophage-encoded ADP-ribosylating exotoxin (‘diphtheria toxin’), whichcan be treated (e.g. using formaldehyde) to give a toxoid that is nolonger toxic but that remains antigenic and is able to stimulate theproduction of specific anti-toxin antibodies after injection. Diphtheriatoxoids are disclosed in more detail in chapter 13 of reference 1.Preferred diphtheria toxoids are those prepared by formaldehydetreatment. The diphtheria toxoid can be obtained by growing C.diphtheriae in growth medium (e.g. Fenton medium, or Linggoud & Fentonmedium), which may be supplemented with bovine extract, followed byformaldehyde treatment, ultrafiltration and precipitation. The toxoidedmaterial may then be treated by a process comprising sterile filtrationand/or dialysis.

A composition should include enough diphtheria toxoid to elicitcirculating diphtheria antitoxin levels of at least 0.01 IU/ml.Quantities of diphtheria toxoid are generally measured in the ‘Lf’ unit(“flocculating units”, or the “limes flocculating dose”, or the “limitof flocculation”), defined as the amount of toxin/toxoid which, whenmixed with one International Unit of antitoxin, produces an optimallyflocculating mixture [27,28]. For example, the NIBSC supplies‘Diphtheria Toxoid, Plain’ [29], which contains 300 LF per ampoule, andalso supplies ‘The 1st International Reference Reagent For DiphtheriaToxoid For Flocculation Test’ [30] which contains 900 Lf per ampoule.The concentration of diphtheria toxoid in a composition can readily bedetermined using a flocculation assay by comparison with a referencematerial calibrated against such reference reagents.

The immunizing potency of diphtheria toxoid in a composition isgenerally expressed in international units (IU). The potency can beassessed by comparing the protection afforded by a composition inlaboratory animals (typically guinea pigs) with a reference vaccine thathas been calibrated in IUs. NIBSC supplies the ‘Diphtheria ToxoidAdsorbed Third International Standard 1999’ [31,32], which contains 160IU per ampoule, and is suitable for calibrating such assays.

The conversion between IU and Lf systems depends on the particulartoxoid preparation.

Compositions of the invention typically include, per unit dose, between1-40 Lf diphtheria toxoid. In a pediatric-type composition, where thediphtheria toxoid is present in an excess relative to tetanus toxoid (inLf units), the composition will generally include between 10-35 Lfdiphtheria toxoid per unit dose e.g. between 15-30 Lf, such as 15, 25 or30 Lf. In a booster-type composition, where tetanus toxoid is present inan excess relative to the diphtheria toxoid (in Lf units), thecomposition will generally include between 1-4 Lf diphtheria toxoid perunit dose e.g. between 1.5-3 Lf, such as 2 or 2.5 Lf. If a compositionincludes saccharide antigen(s) conjugated to diphtheria toxoid thenthese amounts exclude the amount of carrier protein in thoseconjugate(s).

By IU measurements, pediatric-type compositions will generally include≧25 IU diphtheria toxoid per unit dose, whereas booster-typecompositions will generally include 1-3 IU per unit dose.

If a composition includes an aluminium salt adjuvant then diphtheriatoxoid in the composition is preferably adsorbed (more preferablytotally adsorbed) onto it, and preferably onto an aluminium hydroxideadjuvant.

Tetanus Toxoid

Tetanus is caused by Clostridium tetani, a Gram-positive, spore-formingbacillus. This organism expresses an endopeptidase (‘tetanus toxin’),which can be treated to give a toxoid that is no longer toxic but thatremains antigenic and is able to stimulate the production of specificanti-toxin antibodies after injection. Tetanus toxoids are disclosed inmore detail in chapter 27 of reference 1. Preferred tetanus toxoids arethose prepared by formaldehyde treatment. The tetanus toxoid can beobtained by growing C. tetani in growth medium (e.g. a Latham mediumderived from bovine casein), followed by fobrmaldehyde treatment,ultrafiltration and precipitation. The material may then be treated by aprocess comprising sterile filtration and/or dialysis.

A composition should include enough tetanus toxoid to elicit circulatingtetanus antitoxin levels of at least 0.01 IU/ml. Quantities of tetanustoxoid are generally expressed in ‘Lf’ units (see above), defined as theamount of toxoid which, when mixed with one International Unit ofantitoxin, produces an optimally flocculating mixture [27]. The NIBSCsupplies ‘The 1st International Reference Reagent for Tetanus Toxoid ForFlocculation Test’ [33] which contains 1000 LF per ampoule, by whichmeasurements can be calibrated.

The immunizing potency of tetanus toxoid is measured in internationalunits (IU), assessed by comparing the protection afforded by acomposition in laboratory animals (typically guinea pigs) with areference vaccine e.g. using NIBSC's ‘Tetanus Toxoid Adsorbed ThirdInternational Standard 2000’ [34,35], which contains 469 IU per ampoule.

The conversion between IU and Lf systems depends on the particulartoxoid preparation.

Compositions of the invention typically include between 2.5-25 Lf oftetanus toxoid per unit dose. In a pediatric-type composition, wherediphtheria toxoid is present in an excess relative to the tetanus toxoid(in Lf units), the composition will generally include between 4-15 Lftetanus toxoid per unit dose e.g. between 5-10 Lf, such as 5 or 10 Lf.In a booster-type composition, where the tetanus toxoid is present in anexcess relative to diphtheria toxoid (in Lf units), the composition willgenerally include between 4-6 Lf tetanus toxoid per unit dose e.g. 5 Lf.If a composition includes saccharide antigen(s) conjugated to tetanustoxoid then these amounts exclude the amount of carrier protein in thoseconjugate(s).

By IU measurements, pediatric-type compositions will generally include≧40 IU tetanus toxoid per unit dose, whereas booster-type compositionswill generally include 15-25 IU per unit dose.

If a composition includes an aluminium salt adjuvant then tetanus toxoidin the composition is preferably adsorbed (sometimes totally adsorbed)onto an aluminium salt, preferably onto an aluminium hydroxide adjuvant.

Pertussis Toxoid

Bordetella pertussis causes whooping cough. Compositions of theinvention include pertussis toxoid (‘PT’) i.e. a detoxified form ofpertussis toxin. The invention can use a PT-containing whole-cellpertussis antigen (“wP”) but preferably a composition is free from wPand instead includes an acellular (“aP”) PT-containing antigen i.e. adefined mixture of purified pertussis antigens. When using an aP antigena composition of the invention will typically include, in addition tothe PT, filamentous hemagglutinin (FHA) and/or pertactin (also known asthe ‘69 kiloDalton outer membrane protein’). It can also optionallyinclude fimbriae types 2 and 3. Preparation of these various Pa antigensis well known in the art.

PT can be detoxified by treatment with formaldehyde and/orglutaraldehyde, and FHA and pertactin can also be treated in the sameway. As an alternative to chemical detoxification of PT, the inventioncan use a mutant PT in which wild-type enzymatic activity has beenreduced by mutagenesis [36] e.g. the 9K/129G double mutant [37]. The useof such genetically-detoxified PT is preferred,

Quantities of acellular pertussis antigens are usually expressed inmicrograms. Compositions of the invention typically include between 2-30μg PT per unit dose. In a pediatric-type composition, PT can be presentat between 5-30 μg per unit dose (e.g. 5, 7.5, 20 or 25 μg), whereas ina booster-type composition the composition will generally includebetween 2-0 μg PT per unit dose (e.g. 2.5 μg or 8 μg). Where acomposition includes FHA, it is typically present between 2-30 μg perunit dose. In a pediatric-type composition, FHA can be present atbetween 2.5-25 μg per unit dose (e.g. 2.5, 5, 10, 20 or 25 μg), whereasin a booster-type composition FHA can be present at between 4-10 μg perunit dose (e.g. 5 μg or 8 μg). Where a composition includes pertactin,this is typically present between 2-10 μg per unit dose. In apediatric-type composition, pertactin can be present at between 2.5-10μg per unit dose (e.g. 2.5, 3, 8 or 10 μg), whereas in a booster-typecomposition pertactin can be present at between 2-3 μg per unit dose(e.g. 2.5 μg or 3 μg).

A composition normally contains ≦80 μg per unit dose of total acellularpertussis antigens. Each individual antigen will usually be present at≦30 μg per unit dose.

It is usual that each of PT. FHA and pertactin are present in acomposition of the invention. These may be present at various ratios (bymass), such as PT:FHA:p69 ratios of 20:20:3, 25:25:8, 16:16:5, 5:10:6,or 10:5:3. It is usual to have a mass excess of FHA relative topertactin if both are present.

If a composition includes an aluminium salt adjuvant then PT in thecomposition is preferably adsorbed (sometimes totally adsorbed) onto analuminium salt, preferably onto an aluminium hydroxide adjuvant. Any FHAcan also be adsorbed to the aluminium salt. Any pertactin can beadsorbed to the aluminium salt adjuvant, but the presence of pertactinnormally means that the composition requires the presence of aluminiumhydroxide to ensure stable adsorption [38].

Hib Conjugates

Haemophilus influenzae type b (‘Hib’) causes bacterial meningitis. Hibvaccines are typically based on the capsular saccharide antigen (e.g.chapter 14 of ref. 1), the preparation of which is well documented (e.g.references 39 to 48). The Hib saccharide is conjugated to a carrierprotein in order to enhance its immunogenicity, especially in children.Typical carrier proteins are tetanus toxoid, diphtheria toxoid, theCRM197 derivative of diphtheria toxoid, or the outer membrane proteincomplex from serogroup B meningococcus. Tetanus toxoid is a usefulcarrier, as used in the product commonly referred to as ‘PRP-T’. PRP-Tcan be made by activating a Hib capsular polysaccharide using cyanogenbromide, coupling the activated saccharide to an adipic acid linker(such as (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), typically thehydrochloride salt), and then reacting the linker-saccharide entity witha tetanus toxoid carrier protein. CRM197 is another useful carrier forHib conjugate in compositions of the invention.

The saccharide moiety of the conjugate may comprise full-lengthpolyribosylribitol phosphate (PRP) as prepared from Hib bacteria, and/orfragments of full-length PRP. Conjugates with a saccharide:protein ratio(w/w) of between 1:5 (i.e. excess protein) and 5:1 (i.e. excesssaccharide) may be used e.g. ratios between 1:2 and 5:1 and ratiosbetween 1:1.25 and 1:2.5. In preferred vaccines, however, the weightratio of saccharide to carrier protein is between 1:2.5 and 1:3.5. Invaccines where tetanus toxoid is present both as an antigen and as acarrier protein then the weight ratio of saccharide to carrier proteinin the conjugate may be between 1:0.3 and 1:2 [49]. Administration ofthe Hib conjugate preferably results in an anti-PRP antibodyconcentration of ≧0.15 μg/ml, and more preferably ≧1 μg/ml, and theseare the standard response thresholds.

Quantities of Hib antigens are typically expressed in micrograms ofsaccharide. If a composition of the invention includes a Hib antigenthen a normal quantity per unit dose is between 5-15 μg e.g. 10 μg or 12μg.

If a composition includes an aluminium salt adjuvant then Hib antigencan be adsorbed onto it or can be unadsorbed.

Hepatitis B Virus Surface Antigen

Hepatitis B virus (HBV) is one of the known agents which causes viralhepatitis. The HBV virion consists of an inner core surrounded by anouter protein coat or capsid, and the viral core contains the viral DNAgenome. The major component of the capsid is a protein known as HBVsurface antigen or, more commonly, ‘HBsAg’, which is typically a226-amino acid polypeptide with a molecular weight of ˜24 kDa. Allexisting hepatitis B vaccines contain HBsAg, and when this antigen isadministered to a normal vaccinee it stimulates the production ofanti-HBsAg antibodies which protect against HBV infection.

For vaccine manufacture, HBsAg can be made in two ways. The first methodinvolves purifying the antigen in particulate form from the plasma ofchronic hepatitis B carriers, as large quantities of HBsAg aresynthesized in the liver and released into the blood stream during anHBV infection. The second way involves expressing the protein byrecombinant DNA methods. HBsAg for use with the method of the inventionis recombinantly expressed e.g. in yeast or CHO cells. Suitable yeastsinclude Saccharomyces (such as S. cerevisiae) or Hanensula (such as H.polymorpha) hosts.

Unlike native HBsAg (i.e. as in the plasma-purified product),yeast-expressed HBsAg is generally non-glycosylated, and this is themost preferred form of HBsAg for use with the invention. Yeast-expressedHBsAg is highly immunogenic and can be prepared without the risk ofblood product contamination.

The HBsAg will generally be in the form of substantially-sphericalparticles (average diameter of about 20 nm), including a lipid matrixcomprising phospholipids. Yeast-expressed HBsAg particles may includephosphatidylinositol, which is not found in natural HBV virions. Theparticles may also include a non-toxic amount of LPS in order tostimulate the immune system [50]. The particles may retain non-ionicsurfactant (e.g. polysorbate 20) if this was used during disruption ofyeast [51].

A preferred method for HBsAg purification involves, after celldisruption: ultrafiltration; size exclusion chromatography; anionexchange chromatography; ultracentrifugation; desalting; and sterilefiltration. Lysates may be precipitated after cell disruption (e.g.using a polyethylene glycol), leaving HBsAg in solution, ready forultrafiltration.

After purification HBsAg may be subjected to dialysis (e.g. withcysteine), which can be used to remove any mercurial preservatives suchas thimerosal that may have been used during HBsAg preparation [52].Thimerosal-free preparation is preferred.

The HBsAg is preferably from HBV subtype adw2.

Quantities of HBsAg are typically expressed in micrograms. If acomposition of the invention includes HBsAg then a normal quantity perunit dose is between 5-25 μg e.g. 10 μg or 20 μg.

If a composition includes an aluminium salt adjuvant then HBsAg can beadsorbed onto it (preferably adsorbed onto an aluminium phosphateadjuvant).

Inactivated Poliovirus Antigen (IPV)

Poliomyelitis can be caused by one of three types of poliovirus. Thethree types are similar and cause identical symptoms, but they areantigenically very different and infection by one type does not protectagainst infection by others. As explained in chapter 24 of reference 1,it is therefore preferred to use three poliovirus antigens with theinvention—poliovirus Type 1 (e.g. Mahoney strain), poliovirus Type 2(e.g. MEF-1 strain), and poliovirus Type 3 (e.g. Saukett strain). As analternative to these strains (“Salk” strains), Sabin strains of types 1to 3 can be used e.g. as discussed in references 53 & 54. These strainscan be more potent than the normal Salk strains.

Polioviruses may be grown in cell culture. A preferred culture uses aVero cell line, which is a continuous cell line derived from monkeykidney. Vero cells can conveniently be cultured microcarriers. Cultureof the Vero cells before and during viral infection may involve the useof bovine-derived material, such as calf serum, and of lactalbuminhydrolysate (e.g. obtained by enzymatic degradation of lactalbumin).Such bovine-derived material should be obtained from sources which arefree from BSE or other TSEs.

After growth, virions may be purified using techniques such asultrafiltration, diafiltration, and chromatography. Prior toadministration to patients, polioviruses must be inactivated, and thiscan be achieved by treatment with formaldehyde before the viruses areused in the process of the invention.

The viruses are preferably grown, purified and inactivated individually,and are then combined to give a bulk mixture for use with the invention.

Quantities of IPV are typically expressed in the ‘DU’ unit (the“D-antigen unit” [55]). Where all three of Types 1, 2 and 3 poliovirusare present the three antigens can be present at a DU ratio of 5:1:4respectively, or at any other suitable ratio e.g. a ratio of 15:32:45when using Sabin strains [53]. Typical amounts of Salk IPV strains perunit dose are 40 DU type 1, 8 DU type 2 and 32 DU type 3, although lowerdoses can also be used. A low amount of antigen from Sabin strains isparticularly useful, with ≦15 DU type 1, ≦5 DU type 2, and ≦25 DU type 3(per unit dose).

If a composition includes an aluminium salt adjuvant then IPV antigensare often not pre-adsorbed to any adjuvant before they are formulated,but after formulation they may become adsorbed onto the aluminiumsalt(s).

Further Antigens

Compositions of the invention include D, T, and P antigens. As mentionedabove, they may also include Hib, HBsAg, and/or poliovirus antigens.Immunogenic compositions of the invention may include antigens fromfurther pathogens. For example, these antigens may be from N.meningitidis (one or more of serogroups A, B, C, W135 and/or Y) or S.pneumoniae.

Meningococcal Saccharides

Where a composition includes a Neisseria meningitidis capsularsaccharide conjugate there may be one or more than one such conjugate.Including 2, 3, or 4 of serogroups A, C, W135 and Y is typical e.g. A+C,A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135, A+C+Y, A+W135+Y, A+C+W135+Y,etc. Components including saccharides from all four of serogroups A, C,W135 and Y are useful, as in the MENACTRA™ and MENVEO™ products. Whereconjugates from more than one serogroup are included then they may bepresent at substantially equal masses e.g. the mass of each serogroup'ssaccharide is within +10% of each other. A typical quantity perserogroup is between 1 μg and 20 μg e.g. between 2 and 10 μg perserogroup, or about 4 μg or about 5 μg or about 10 μg. As an alternativeto a substantially equal ratio, a double mass of serogroup A saccharidemay be used.

Administration of a conjugate preferably results in an increase in serumbactericidal assay (SBA) titre for the relevant serogroup of at least4-fold, and preferably at least 8-fold. SBA titres can be measured usingbaby rabbit complement or human complement [56].

The capsular saccharide of serogroup A meningococcus is a homopolymer of(αl→6)-linked N-acetyl-D-mannosamine-1-phosphate, with partialO-acetylation in the C3 and C4 positions. Acetylation at the C-3position can be 70-95%. Conditions used to purify the saccharide canresult in de-O-acetylation (e.g. under basic conditions), but it isuseful to retain OAc at this C-3 position. In some embodiments, at least50% (e.g. at least 60%, 70%, 80%, 90%, 95% or more) of the mannosamineresidues in a serogroup A saccharides are O-acetylated at the C-3position. Acetyl groups can be replaced with blocking groups to preventhydrolysis [57], and such modified saccharides are still serogroup Asaccharides within the meaning of the invention.

The serogroup C capsular saccharide is a homopolymer of (α2→9)-linkedsialic acid (N-acetyl neuraminic acid, or ‘NeuNAc’). The saccharidestructure is written as →9)-Neu p NAc 7/8 OAc-(α2→. Most serogroup Cstrains have O-acetyl groups at C-7 and/or C-8 of the sialic acidresidues, but about 15% of clinical isolates lack these O-acetyl groups[58,59]. The presence or absence of OAc groups generates uniqueepitopes, and the specificity of antibody binding to the saccharide mayaffect its bactericidal activity against O-acetylated (OAc−) andde-O-acetylated (OAc+) strains [60-62]. Serogroup C saccharides usedwith the invention may be prepared from either OAc+ or OAc− strains.Licensed MenC conjugate vaccines include both OAc− (NEISVAC-C™) and OAc+(MENJUGATE™ & MENINGITEC™) saccharides. In some embodiments, strains forproduction of serogroup C conjugates are OAc+ strains, e.g. of serotype16, serosubtype P1.7a,1, etc. Thus C:16:P1.7a,1 OAc+ strains may beused. OAc+ strains in serosubtype P1.1 are also useful, such as the C11strain. Preferred MenC saccharides are taken from OAc+ strains, such asstrain C11.

The serogroup W135 saccharide is a polymer of sialic acid-galactosedisaccharide units. Like the serogroup C saccharide, it has variableO-acetylation, but at sialic acid 7 and 9 positions [63]. The structureis written as: →4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Gal-α-(1→.

The serogroup Y saccharide is similar to the serogroup W135 saccharide,except that the disaccharide repeating unit includes glucose instead ofgalactose. Like serogroup W135, it has variable O-acetylation at sialicacid 7 and 9 positions [63]. The serogroup Y structure is written as:→4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Glc-α-(1→.

The saccharides used according to the invention may be O-acetylated asdescribed above (e.g. with the same O-acetylation pattern as seen innative capsular saccharides), or they may be partially or totallyde-O-acetylated at one or more positions of the saccharide rings, orthey may be hyper-O-acetylated relative to the native capsularsaccharides. For example, reference 64 reports the use of serogroup Ysaccharides that are more than 80% de-O-acetylated.

The saccharide moieties in meningococcal conjugates may comprisefull-length saccharides as prepared from meningococci, and/or maycomprise fragments of full-length saccharides i.e. the saccharides maybe shorter than the native capsular saccharides seen in bacteria. Thesaccharides may thus be depolymerised, with depolymerisation occurringduring or after saccharide purification but before conjugation.Depolymerisation reduces the chain length of the saccharides. Onedepolymerisation method involves the use of hydrogen peroxide [65].Hydrogen peroxide is added to a saccharide (e.g. to give a final H₂O₂concentration of 1%), and the mixture is then incubated (e.g. at about55° C.) until a desired chain length reduction has been achieved.Another depolymerisation method involves acid hydrolysis [66]. Otherdepolymerisation methods are known in the art. The saccharides used toprepare conjugates for use according to the invention may be obtainableby any of these depolymerisation methods. Depolymerisation can be usedin order to provide an optimum chain length for immunogenicity and/or toreduce chain length for physical manageability of the saccharides. Insome embodiments, saccharides have the following range of averagedegrees of polymerisation (Dp): A=10-20; C=12-22; W135=15-25; Y=15-25.In terms of molecular weight, rather than Dp, useful ranges are, for allserogroups: <100 kDa; 5 kDa-75 kDa; 7 kDa-50 kDa; 8 kDa-35 kDa; 12kDa-25 kDa; 15 kDa-22 kDa. In other embodiments, the average molecularweight for saccharides from each of meningococcal serogroups A, C, W135and Y may be more than 50 kDa e.g. ≧75 kDa, ≧100 kDa, ≧110 kDa, ≧120kDa, ≧130 kDa, etc. [67], and even up to 1500 kDa, in particular asdetermined by MALLS. For instance: a MenA saccharide may be in the range50-500 kDa e.g. 60-80 kDa; a MenC saccharide may be in the range 100-210kDa; a MenW135 saccharide may be in the range 60-190 kDa e.g. 120-140kDa; and/or a MenY saccharide may be in the range 60-190 kDa e.g.150-160 kDa.

If a component or composition includes both Hib and meningococcalconjugates then, in some embodiments, the mass of Hib saccharide can besubstantially the same as the mass of a particular meningococcalserogroup saccharide. In some embodiments, the mass of Hib saccharidewill be more than (e.g. at least 1.5×) the mass of a particularmeningococcal serogroup saccharide. In some embodiments, the mass of Hibsaccharide will be less than (e.g. at least 1.5× less) the mass of aparticular meningococcal serogroup saccharide.

Where a composition includes saccharide from more than one meningococcalserogroup, there is an mean saccharide mass per serogroup. Ifsubstantially equal masses of each serogroup are used then the mean masswill be the same as each individual mass; where non-equal masses areused then the mean will differ e.g. with a 10:5:5:5 μg amount for aMenACWY mixture, the mean mass is 6.25 μg per serogroup. In someembodiments, the mass of Hib saccharide will be substantially the sameas the mean mass of meningococcal saccharide per serogroup. In someembodiments, the mass of Hib saccharide will be more than (e.g. at least1.5×) the mean mass of meningococcal saccharide per serogroup. In someembodiments, the mass of Hib saccharide will be less than (e.g. at least1.5×) the mean mass of meningococcal saccharide per serogroup [68].

Pneumococcal Saccharides

Streptococcus pneumoniae causes bacterial meningitis and existingvaccines are based on capsular saccharides. Thus compositions of theinvention can include at least one pneumococcal capsular saccharideconjugated to a carrier protein.

The invention can include capsular saccharide from one or more differentpneumococcal serotypes. Where a composition includes saccharide antigensfrom more than one serotype, these are preferably prepared separately,conjugated separately, and then combined. Methods for purifyingpneumococcal capsular saccharides are known in the art (e.g. seereference 69) and vaccines based on purified saccharides from 23different serotypes have been known for many years. Improvements tothese methods have also been described e.g. for serotype 3 as describedin reference 70, or for serotypes 1, 4, 5, 6A, 6B, 7F and 19A asdescribed in reference 71.

Pneumococcal capsular saccharide(s) will typically be selected from thefollowing serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F. Thus, intotal, a composition may include a capsular saccharide from 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 ormore different serotypes. Compositions which include at least serotype6B saccharide are useful.

A useful combination of serotypes is a 7-valent combination e.g.including capsular saccharide from each of serotypes 4, 6B, 9V, 14, 18C,19F, and 23F. Another useful combination is a 9-valent combination e.g.including capsular saccharide from each of serotypes 1, 4, 5, 6B, 9V,14, 18C, 19F and 23F. Another useful combination is a 10-valentcombination e.g. including capsular saccharide from each of serotypes 1,4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valent combination mayfurther include saccharide from serotype 3. A 12-valent combination mayadd to the 10-valent mixture: serotypes 6A and 19A; 6A and 22F; 19A and22F; 6A and 15B; 19A and 15B; or 22F and 15B. A 13-valent combinationmay add to the 11-valent mixture: serotypes 19A and 22F; 8 and 12F; 8and 15B; 8 and 19A; 8 and 22F; 12F and 15B; 12F and 19A; 12F and 22F;15B and 19A; 15B and 22F; 6A and 19A, etc.

Thus a useful 13-valent combination includes capsular saccharide fromserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19 (or 19A), 19F and 23Fe.g. prepared as disclosed in references 72 to 75. One such combinationincludes serotype 6B saccharide at about 8 μg/ml and the other 12saccharides at concentrations of about 4 μg/ml each. Another suchcombination includes serotype 6A and 6B saccharides at about 8 μg/mleach and the other 11 saccharides at about 4±g/ml each.

Suitable carrier proteins for conjugates include bacterial toxins, suchas diphtheria or tetanus toxins, or toxoids or mutants thereof. Theseare commonly used in conjugate vaccines. For example, the CRM197diphtheria toxin mutant is useful [76]. Other suitable carrier proteinsinclude synthetic peptides [77,78], heat shock proteins [79,80],pertussis proteins [81,82], cytokines [83], lymphokines [83], hormones[83], growth factors [83], artificial proteins comprising multiple humanCD4⁺ T cell epitopes from various pathogen-derived antigens [84] such asN19 [85], protein D from H. influenzae [86-88], pneumolysin [89] or itsnon-toxic derivatives [90], pneumococcal surface protein PspA [91],iron-uptake proteins [92], toxin A or B from C. difficile [93],recombinant Pseudomonas aeruginosa exoprotein A (rEPA) [94], etc.

Particularly useful carrier proteins for pneumococcal conjugate vaccinesare CRM197, tetanus toxoid, diphtheria toxoid and H. influenzae proteinD. CRM197 is used in PREVNAR™. A 13-valent mixture may use CRM197 as thecarrier protein for each of the 13 conjugates, and CRM197 may be presentat about 55-60 μg/ml.

Where a composition includes conjugates from more than one pneumococcalserotype, it is possible to use the same carrier protein for eachseparate conjugate, or to use different carrier proteins. In both cases,though, a mixture of different conjugates will usually be formed bypreparing each serotype conjugate separately, and then mixing them toform a mixture of separate conjugates. Reference 95 describes potentialadvantages when using different carrier proteins in multivalentpneumococcal conjugate vaccines, but the PREVNAR™ product successfullyuses the same carrier for each of seven different serotypes.

A carrier protein may be covalently conjugated to a pneumococcalsaccharide directly or via a linker. Various linkers are known. Forexample, attachment may be via a carbonyl, which may be formed byreaction of a free hydroxyl group of a modified saccharide with CDI[96.97]followed by reaction with a protein to form a carbamate linkage.Carbodiimide condensation can be used [98]. An adipic acid linker can beused, which may be formed by coupling a free —NH₂ group (e.g. introducedto a saccharide by amination) with adipic acid (using, for example,diimide activation), and then coupling a protein to the resultingsaccharide-adipic acid intermediate [99,100]. Other linkers includeβ-propionamido [101], nitrophenyl-ethylamine [102], haloacyl halides[103], glycosidic linkages [104], 6-aminocaproic acid [105],N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) [106], adipic aciddihydrazide ADH [107], C₄ to C₁₂ moieties [108], etc.

Conjugation via reductive amination can be used. The saccharide mayfirst be oxidised with periodate to introduce an aldehyde group whichcan then form a direct covalent linkage to a carrier protein byreductive amination e.g. to a lysine's ε-amino group. If the saccharideincludes multiple aldehyde groups per molecule then this linkagetechnique can lead to a cross-linked product, where multiple aldehydesreact with multiple carrier amines. This cross-linking conjugationtechnique is particularly useful for at least pneumococcal serotypes 4,6B, 9V, 14, 18C, 19F and 23F.

A pneumococcal saccharide may comprise a full-length intact saccharideas prepared from pneumococcus, and/or may comprise fragments offull-length saccharides i.e. the saccharides may be shorter than thenative capsular saccharides seen in bacteria. The saccharides may thusbe depolymerised, with depolymerisation occurring during or aftersaccharide purification but before conjugation. Depolymerisation reducesthe chain length of the saccharides.

Depolymerisation can be used in order to provide an optimum chain lengthfor immunogenicity and/or to reduce chain length for physicalmanageability of the saccharides. Where more than one pneumococcalserotype is used then it is possible to use intact saccharides for eachserotype, fragments for each serotype, or to use intact saccharides forsome serotypes and fragments for other serotypes.

Where a composition includes saccharide from any of serotypes 4, 6B, 9V,14, 19F and 23F, these saccharides are preferably intact. In contrast,where a composition includes saccharide from serotype 18C, thissaccharide is preferably depolymerised.

A serotype 3 saccharide may also be depolymerised, For instance, aserotype 3 saccharide can be subjected to acid hydrolysis fordepolymerisation [72] e.g. using acetic acid. The resulting fragmentsmay then be oxidised for activation (e.g. periodate oxidation, maybe inthe presence of bivalent cations e.g. with MgCl₂), conjugated to acarrier (e.g. CRM197) under reducing conditions (e.g. using sodiumcyanoborohydride), and then (optionally) any unreacted aldehydes in thesaccharide can be capped (e.g. using sodium borohydride) [72].Conjugation may be performed on lyophilized material e.g. afterco-lyophilizing activated saccharide and carrier. A serotype 1saccharide may be at least partially de-O-acetylated e.g. achieved byalkaline pH buffer treatment [73] such as by using abicarbonate/carbonate buffer. Such (partially) de-O-acetylatedsaccharides can be oxidised for activation (e.g. periodate oxidation),conjugated to a carrier (e.g. CRM197) under reducing conditions (e.g.using sodium cyanoborohydride), and then (optionally) any unreactedaldehydes in the saccharide can be capped (e.g. using sodiumborohydride) [73]. Conjugation may be performed on lyophilized materiale.g. after co-lyophilizing activated saccharide and carrier.

A serotype 19A saccharide may be oxidised for activation (e.g. periodateoxidation), conjugated to a carrier (e.g. CRM197) in DMSO under reducingconditions, and then (optionally) any unreacted aldehydes in thesaccharide can be capped (e.g. using sodium borohydride) [109].Conjugation may be performed on lyophilized material e.g. afterco-lyophilizing activated saccharide and carrier.

One or more pneumococcal capsular saccharide conjugates may be presentin lyophilised form.

Pneumococcal conjugates can ideally elicit anticapsular antibodies thatbind to the relevant saccharide e.g. elicit an anti-saccharide antibodylevel ≧0.20 μg/mL [110]. The antibodies may be evaluated by enzymeimmunoassay (EIA) and/or measurement of opsonophagocytic activity (OPA).The EIA method has been extensively validated and there is a linkbetween antibody concentration and vaccine efficacy.

Adjuvants

Compositions of the invention can include an adjuvant, such as (i) anoil-in-water emulsion (ii) at least one aluminium salt or (iii) at leastone TLR agonist.

In some embodiments a composition includes a mixture of an aluminiumsalt and a TLR agonist, and the TLR agonist can be adsorbed to thealuminium salt to improve adjuvant effects [142]. This can lead to abetter (stronger, or more quickly achieved) immune response and/or canpermit a reduction in the amount of aluminium in the composition whilemaintaining an equivalent adjuvant effect.

Where a composition includes aluminium salt adjuvant(s) then between oneand all of the immunogens in the composition can be adsorbed to thesalt(s). Moreover, if the composition includes a TLR adjuvant then thiscan also be adsorbed to the salt(s), as discussed below.

Where a composition includes an aluminium salt adjuvant then preferablyit does not also include an oil-in-water emulsion adjuvant. Conversely,where a composition includes an oil-in-water emulsion adjuvant thenpreferably it does not also include an aluminium salt adjuvant.

Oil-in-Water Emulsion Adjuvants

According to the invention's second aspect a vaccine is adjuvanted withan oil-in-water emulsion. Various such emulsions are known e.g. MF59 andAS03 are both authorised in Europe.

Useful emulsion adjuvants they typically include at least one oil and atleast one surfactant, with the oil(s) and surfactant(s) beingbiodegradable (metabolisable) and biocompatible. The oil droplets in theemulsion generally have a sub-micron diameter, and these small sizes canreadily be achieved with a microfluidiser to provide stable emulsions,or by alternative methods e.g. phase inversion. Emulsions in which atleast 80% (by number) of droplets have a diameter of less than 220 nmare preferred, as they can be subjected to filter sterilization.

The emulsion can include oil(s) from an animal (such as fish) and/orvegetable source. Sources for vegetable oils include nuts, seeds andgrains. Peanut oil, soybean oil, coconut oil, and olive oil, the mostcommonly available, exemplify the nut oils. Jojoba oil can be used e.g.obtained from the jojoba bean. Seed oils include safflower oil,cottonseed oil, sunflower seed oil, sesame seed oil and the like. In thegrain group, corn oil is the most readily available, but the oil ofother cereal grains such as wheat, oats, rye, rice, teff, triticale andthe like may also be used. 6-10 carbon fatty acid esters of glycerol and1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolisable and may therefore be used with theinvention. The procedures for separation, purification, saponificationand other means necessary for obtaining pure oils from animal sourcesare well known in the art.

Most fish contain metabolisable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Shark liver oil contains a branched, unsaturated terpenoids known assqualene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene,which is particularly preferred for use with the invention (see below).Squalane, the saturated analog to squalene, is also a useful oil. Fishoils, including squalene and squalane, are readily available fromcommercial sources or may be obtained by methods known in the art. Otherpreferred oils are the tocopherols (see below). Mixtures of oils can beused.

Preferred amounts of total oil (% by volume) in an adjuvant emulsion arebetween 1 and 20% e.g. between 2-10%. A squalane content of 5% by volumeis particularly useful.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10 e.g. about 15. The invention can be used with surfactants including,but not limited to: the polyoxyethylene sorbitan esters surfactants(commonly referred to as the Tweens), especially polysorbate 20 orpolysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO),and/or butylene oxide (BO), sold under the DOWFAX™ tradename, such aslinear EO/PO block copolymers; octoxynols, which can vary in the numberof repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9(Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particularinterest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40);phospholipids such as phosphatidylcholine (lecithin); nonylphenolethoxylates, such as the Tergitol™ NP series; polyoxyethylene fattyethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known asBrij surfactants), such as triethyleneglycol monolauryl ether (Brij 30);and sorbitan esters (commonly known as the Spans), such as sorbitantrioleate (Span 85) or sorbitan monolaurate.

Emulsions used with the invention preferably include non-ionicsurfactant(s). Preferred surfactants for including in the emulsion arepolysorbate 80 (polyoxyethylene sorbitan monooleate; Tween 80), Span 85(sorbitan trioleate), lecithin or Triton X-100. Mixtures of surfactantscan be used e.g. a mixture of polysorbate 80 and sorbitan trioleate. Acombination of a polyoxyethylene sorbitan ester such as polysorbate 80(Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol(Triton X-100) is also useful. Another useful combination compriseslaureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.Where a mixture of surfactants is used then the HLB of the mixture iscalculated according to their relative weightings (by volume) e.g. thepreferred 1:1 mixture by volume of polysorbate 80 and sorbitan trioleatehas a HLB of 8.4.

Preferred amounts of total surfactant (% by volume) in an adjuvantemulsion are between 0.1 and 2% e.g. between 0.25-2%. A total content of1% by volume is particularly useful e.g. 0.5% by volume of polysorbate80 and 0.5% by volume of sorbitan trioleate.

Useful emulsions can be prepared using known techniques e.g. seereferences 132 and 111-112117

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, polysorbate 80, and sorbitan        trioleate. The composition of the emulsion by volume can be        about 5% squalene, about 0.5% polysorbate 80 and about 0.5%        sorbitan trioleate. In weight terms, these ratios become 4.3%        squalene, 0.5% polysorbate 80 and 0.48% sorbitan trioleate. This        adjuvant is known as ‘MF59’ [118-120], as described in more        detail in Chapter 10 of ref. 131 and chapter 12 of ref. 132. The        MF59 emulsion advantageously includes citrate ions e.g. 10 mM        sodium citrate buffer.    -   An emulsion of squalene, a tocopherol, and polysorbate 80. The        emulsion may include phosphate buffered saline. These emulsions        may have from 2 to 10% squalene, from 2 to 10% tocopherol and        from 0.3 to 3% polysorbate 80, and the weight ratio of        squalene:tocopherol is preferably ≦1 (e.g. 0.90) as this can        provide a more stable emulsion. Squalene and polysorbate 80 may        be present volume ratio of about 5:2, or at a weight ratio of        about 11:5. Thus the three components (squalene, tocopherol,        polysorbate 80) may be present at a weight ratio of        1068:1186:485 or around 55:61:25. This adjuvant is known as        ‘AS03’. Another useful emulsion of this type may comprise, per        human dose, 0.5-10 mg squalene, 0.5-11 mg tocopherol, and 0.1-4        mg polysorbate 80 [121] e.g. in the ratios discussed above.    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [122].    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 123, preferred phospholipid components        are phosphatidylcholine, phosphatidyl ethanolamine,        phosphatidylserine, phosphatidylinositol phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [124]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. It may also        include a TLR4 agonist, such as one whose chemical structure        does not include a sugar ring [125]. Such emulsions may be        lyophilized. The ‘AF03’ product is one such emulsion.

Preferred oil-in-water emulsions used with the invention comprisesqualene and polysorbate 80.

The emulsions may be mixed with TdaP antigens during vaccinemanufacture, or they may be mixed extemporaneously at the time ofdelivery. Thus, in some embodiments, the adjuvant and antigens may bekept separately in a packaged or distributed vaccine, ready for finalformulation at the time of use. At the time of mixing (whether duringbulk manufacture, or at the point of use) the antigen will generally bein an aqueous form, such that the final vaccine is prepared by mixingtwo liquids. The volume ratio of the two liquids for mixing can vary(e.g. between 5:1 and 1:5) but is generally about 1:1. If emulsion andantigen are stored separately in a kit then the product may be presentedas a vial containing emulsion and a vial containing aqueous antigen, formixing to give adjuvanted liquid vaccine (monodose or multi-dose).

Preferred emulsions of the invention include squalene oil. This isusually prepared from shark oil but alternative sources are known e.g.see references 126 (yeast) and 127 (olive oil). Squalene which containsless than 661 picograms of PCBs per gram of squalene (TEQ) is preferredfor use with the invention, as disclosed in reference 128. The emulsionsare preferably made from squalene of high purity e.g. prepared bydouble-distillation as disclosed in reference 129.

Where a composition includes a tocopherol, any of the a, 03, y, 8, c or4 tocopherols can be used, but α-tocopherols are preferred. Thetocopherol can take several forms e.g. different salts and/or isomers.Salts include organic salts, such as succinate, acetate, nicotinate,etc. D-α-tocopherol and DL-α-tocopherol can both be used. Tocopherolshave antioxidant properties that may help to stabilize the emulsions[130]. A preferred α-tocopherol is DL-α-tocopherol, and a preferred saltof this tocopherol is the succinate.

Aluminium Salt Adjuvants

Compositions of the invention can include an aluminium salt adjuvant.Aluminium salt adjuvants currently in use are typically referred toeither as “aluminium hydroxide” or as “aluminium phosphate” adjuvants.These are names of convenience, however, as neither is a precisedescription of the actual chemical compound which is present (e.g. seechapter 9 of reference 131, and chapter 4 of reference 132). Theinvention can use any of the “hydroxide” or “phosphate” salts thatuseful as adjuvants. Aluminium salts which include hydroxide ions arepreferred if adsorption of a TLR agonist is desired as these hydroxideions can readily undergo ligand exchange for adsorption of the TLRagonist. Thus preferred salts for adsorption of TLR agonists arealuminium hydroxide and/or aluminium hydroxyphosphate. These havesurface hydroxyl moieties which can readily undergo ligand exchange withphosphorus-containing groups (e.g. phosphates, phosphonates) to providestable adsorption. An aluminium hydroxide adjuvant is thus mostpreferred.

The adjuvants known as “aluminium hydroxide” are typically aluminiumoxyhydroxide salts, which are usually at least partially crystalline.Aluminium oxyhydroxide, which can be represented by the formula AlO(OH),can be distinguished from other aluminium compounds, such as aluminiumhydroxide Al(OH)₃, by infrared (IR) spectroscopy, in particular by thepresence of an adsorption band at 1070 cm⁻¹ and a strong shoulder at3090-3100 cm⁻¹ (chapter 9 of ref. 131). The degree of crystallinity ofan aluminium hydroxide adjuvant is reflected by the width of thediffraction band at half height (WHH), with poorly-crystalline particlesshowing greater line broadening due to smaller crystallite sizes. Thesurface area increases as WHH increases, and adjuvants with higher WHHvalues have been seen to have greater capacity for antigen adsorption. Afibrous morphology (e.g. as seen in transmission electron micrographs)is typical for aluminium hydroxide adjuvants e.g. with needle-likeparticles with diameters about 2 nm. The PZC of aluminium hydroxideadjuvants is typically about 11 i.e. the adjuvant itself has a positivesurface charge at physiological pH. Adsorptive capacities of between1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported foraluminium hydroxide adjuvants.

The adjuvants known as “aluminium phosphate” are typically aluminiumhydroxyphosphates, often also containing a small amount of sulfate. Theymay be obtained by precipitation, and the reaction conditions andconcentrations during precipitation influence the degree of substitutionof phosphate for hydroxyl in the salt. Hydroxyphosphates generally havea PO₄/Al molar ratio between 0.3 and 0.99. Hydroxyphosphates can bedistinguished from strict AlPO₄ by the presence of hydroxyl groups. Forexample, an IR spectrum band at 3164 cm⁻¹ (e.g. when heated to 200° C.)indicates the presence of structural hydroxyls (chapter 9 of ref. 131).

The PO₄/Al^(j) molar ratio of an aluminium phosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, andmore preferably 0.95±0.1. The aluminium phosphate will generally beamorphous, particularly for hydroxyphosphate salts. A typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. The aluminium phosphate willgenerally be particulate. Typical diameters of the particles are in therange 0.5-20 μm (e.g. about 5-10 μm) after any antigen adsorption.Adsorptive capacities of between 0.7-1.5 mg protein per mg Al⁺⁺⁺ at pH7.4 have been reported for aluminium phosphate adjuvants.

The PZC of aluminium phosphate is inversely related to the degree ofsubstitution of phosphate for hydroxyl, and this degree of substitutioncan vary depending on reaction conditions and concentration of reactantsused for preparing the salt by precipitation. PZC is also altered bychanging the concentration of free phosphate ions in solution (morephosphate=more acidic PZC) or by adding a buffer such as a histidinebuffer (makes PZC more basic). Aluminium phosphates used according tothe invention will generally have a PZC of between 4.0 and 7.0, morepreferably between 5.0 and 6.5 e.g. about 5.7.

In solution both aluminium phosphate and hydroxide adjuvants tend toform stable porous aggregates 1-10 μm in diameter [133].

A composition can include a mixture of both an aluminium hydroxide andan aluminium phosphate, and components may be adsorbed to one or both ofthese salts.

An aluminium phosphate solution used to prepare a composition of theinvention may contain a buffer (e.g. a phosphate or a histidine or aTris buffer), but this is not always necessary. The aluminium phosphatesolution is preferably sterile and pyrogen-free. The aluminium phosphatesolution may include free aqueous phosphate ions e.g. present at aconcentration between 1.0 and 20 mM, preferably between 5 and 15 mM, andmore preferably about 10 mM. The aluminium phosphate solution may alsocomprise sodium chloride. The concentration of sodium chloride ispreferably in the range of 0.1 to 100 mg/ml (e.g. 0.5-50 mg/ml, 1-20mg/ml, 2-10 mg/ml) and is more preferably about 3±1 mg/ml. The presenceof NaCl facilitates the correct measurement of pH prior to adsorption ofantigens.

A composition of the invention ideally includes less than 0.85 mg Al⁺⁺⁺per unit dose. In some embodiments of the invention a compositionincludes less than 0.5 mg Al⁺⁺⁺ per unit dose. The amount of Al⁺⁺⁺ canbe lower than this e.g. <250 μg, <200 μg, <150 μg, <100 μg, <75 Ag, <50μg, <25 μg, <10 μg, etc.

Where compositions of the invention include an aluminium-based adjuvant,settling of components may occur during storage. The composition shouldtherefore be shaken prior to administration to a patient. The shakencomposition will be a turbid white suspension.

If a TLR agonist and an aluminium salt are both present, in general theweight ratio of the TLR agonist to Al^(I I I) will be less than 5:1 e.g.less than 4:1, less than 3:1, less than 2:1, or less than 1:1. Thus, forexample, with an Al⁺⁺⁺ concentration of 0.5 mg/ml the maximumconcentration of TLR agonist would be 2.5 mg/ml. But higher or lowerlevels can be used. A lower mass of TLR agonist than of Al⁺⁺⁺ can bemost typical e.g. per dose, 1000 μg of TLR agonist with 0.2 mg Al⁺⁺⁺,etc. For instance, the FENDRIX™ product includes 50μg of 3d-MPL and 0.5mg Al⁺⁺⁺ per dose.

TLR Agonists

In some embodiments a composition of the invention includes a TLRagonist i.e. a compound which can agonise a Toll-like receptor. Mostpreferably, a TLR agonist is an agonist of a human TLR. The TLR agonistcan activate any of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9or TLR11; preferably it can activate human TLR4 or human TLR7.

Agonist activity of a compound against any particular Toll-like receptorcan be determined by standard assays. Companies such as Imgenex andInvivogen supply cell lines which are stably co-transfected with humanTLR genes and NFκB, plus suitable reporter genes, for measuring TLRactivation pathways. They are designed for sensitivity, broad workingrange dynamics and can be used for high-throughput screening.Constitutive expression of one or two specific TLRs is typical in suchcell lines. See also reference 134. Many TLR agonists are known in theart e.g. reference 135 describes certain lipopeptide molecules that areTLR2 agonists, references 136 to 139 each describe classes of smallmolecule agonists of TLR7, and references 140 & 141 describe TLR7 andTLR8 agonists for treatment of diseases.

A TLR agonist used with the invention ideally includes at least oneadsorptive moiety. The inclusion of such moieties in TLR agonists allowsthem to adsorb to insoluble aluminium salts (e.g. by ligand exchange orany other suitable mechanism) and improves their immunological behaviour[142]. Phosphorus-containing adsorptive moieties are particularlyuseful, and so an adsorptive moiety may comprise a phosphate, aphosphonate, a phosphinate, a phosphonite, a phosphinite, etc.

Preferably the TLR agonist includes at least one phosphonate group.

Thus, in preferred embodiments, a composition of the invention includesa TLR agonist (such as a TLR7 agonist) which includes a phosphonategroup. This phosphonate group can allow adsorption of the agonist to aninsoluble aluminium salt [142].

TLR agonists useful with the invention may include a single adsorptivemoiety, or may include more than one e.g. between 2 and 15 adsorptivemoieties. Typically a compound will include 1, 2 or 3 adsorptivemoieties.

Phosphorus-containing TLR agonists useful with the invention can berepresented by formula (A1):

-   -   wherein:        -   R^(X) and R^(Y) are independently selected from H and C₁-C₆            alkyl;        -   X is selected from a covalent bond, O and NH;        -   Y is selected from a covalent bond, O, C(O), S and NH;        -   L is a linker e.g. selected from, C₁-C₆alkylene,            C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy            and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted            with 1 to 4 substituents independently selected from halo,            OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;        -   each p is independently selected from 1, 2, 3, 4, 5 and 6;        -   q is selected from 1, 2, 3 and 4;        -   n is selected from 1, 2 and 3; and        -   A is a TLR agonist moiety.

In one embodiment, the TLR agonist according to formula (A1) is asfollows: R^(X) and R^(Y) are H; X is O; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substitutedwith 1 to 2 halogen atoms; p is selected from 1, 2 and 3; q is selectedfrom 1 and 2; and n is 1. Thus in these embodiments the adsorptivemoiety comprises a phosphate group.

In other embodiments, the TLR agonist according to formula (A1) is asfollows: R^(X) and R^(Y) are H; X is a covalent bond; L is selected fromC₁-C₆ alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 2 halogen atoms; p is selected from 1, 2 or 3; qis selected from 1 or 2; and n is 1. Thus in these embodiments theadsorptive moiety comprises a phosphonate group.

Useful ‘A’ moieties for formula (A1) include, but are not limited to,radicals of any of the following compounds, defined herein or asdisclosed in references 136, 137, 139, 140, 142 & 177:

In some embodiments, the TLR agonist moiety ‘A’ has a molecular weightof less than 1000 Da. In some embodiments, the TLR agonist of formula(A1) has a molecular weight of less than 1000 Da.

Preferred TLR agonists are water-soluble. Thus they can form ahomogenous solution when mixed in an aqueous buffer with water at pH 7at 25° C. and 1 atmosphere pressure to give a solution which has aconcentration of at least 50 μg/ml. The term “water-soluble” thusexcludes substances that are only sparingly soluble under theseconditions.

Useful TLR agonists include those having formula (C), (D), (E), (F),(G), (H), (I), (II), (J) or (K) as described in more detail below. Otheruseful TLR agonists are compounds 1 to 102 as defined in reference 142.Preferred TLR7 agonists have formula (K), such as compound K2 identifiedbelow. These can be used as salts e.g. the arginine salt of K2.

Preferred TLR4 agonists are analogs of monophosphoryl lipid A (MPL), asdescribed in more detail below. For instance, a useful TLR4 agonist is a3d-MPL.

A composition of the invention can include more than one TLR agonist.These two agonists are different from each other and they can target thesame TLR or different TLRs. Both agonists can be adsorbed to analuminium salt.

It is preferred that at least 50% (by mass) of any TLR agonist(s) in thecomposition is adsorbed to an aluminium salt (if present) e.g. ≧60%,≧70%, ≧80%, ≧85%, ≧90%, ≧92%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, oreven 100%.

Where a composition of the invention includes a TLR agonist adsorbed toa metal salt, and also includes a buffer, it is preferred that theconcentration of any phosphate ions in the buffer should be less than 50mM (e.g. between 1-15 mM) as a high concentration of phosphate ions cancause desorption. Use of a histidine buffer is preferred.

Formulae (C), (D), (E) and (H)—TLR7 Agonists

The TLR agonist can be a compound according to any of formulae (C), (D),(E), and (H):

wherein:

-   -   (a) P³ is selected from H, C₁-C₆alkyl, CF₃, and        —((CH₂)_(p)O)(CH₂)_(p)O_(s)— and —Y-L-X—P(O)(OR^(X))(OR^(Y));        and P⁴ is selected from H, C₁-C₆alkyl, —C₁-C₆alkylaryl and        —Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one        of P³ and P⁴ is —Y-L-X—P(O)(OR^(X))(OR^(Y)),    -   (b) P⁵ is selected from H, C₁-C₆alkyl, and        —Y-L-X—P(OR^(X))(OR^(Y)); P⁶ is selected from H, C₁-C₆alkyl each        optionally substituted with 1 to 3 substituents selected from        C₁-C₄alkyl and OH, and —Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁷ is        selected from H, C₁-C₆alkyl, —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)—,        —NHC₁-C₆alkyl and —Y-L-X—P(OR^(X))(OR^(Y)); with the proviso        that at least one of P⁵, P⁶ and P⁷ is        —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   (c) P⁸ is selected from H. C₁-C₆alkyl, C₁-C₆alkoxy,        —NHC₁-C₆alkyl each optionally substituted with OH, and        —Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁹ and P¹⁰ are each        independently selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,        —NHC₁-C₆alkyl each optionally substituted with OH and        C₁-C₆alkyl, and —Y-L-X—P(OR^(X))(OR^(Y)); with the proviso that        at least one of P⁸, P⁹ or P¹⁰ is —Y-L-X—P(OR^(X))(OR^(Y));    -   (d) P¹⁶ and each P¹⁸ are each independently selected from H,        C₁-C₆alkyl, and —Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹⁷ is selected        from H, C₁-C₆alkyl aryl, heteroaryl, C₁-C₆alkylaryl, C₁-C₆alkyl        heteroaryl, C₁-C₆alkylaryl-Y-L-X—P(OR^(X))(OR^(Y)) and        —Y-L-X—P(O)(OR^(X))(OR^(Y)), each optionally substituted with 1        to 2 substituents selected from C₁-C₆alkyl or heterocyclyl with        the proviso that at least one of P¹⁶, P¹⁷ or a P¹⁸ contains a        —Y-L-X—P(O)(OR^(X))(OR^(Y)) moiety;    -   R^(X) and R^(Y) are independently selected from H and        C₁-C₆alkyl;    -   R^(C), R^(D) and R^(H) are each independently selected from H        and C₁-C₆alkyl;    -   X^(C) is selected from CH and N;    -   R^(E) is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,        C(O)C₁-C₆alkyl, halogen and —((CH₂)_(p)O)_(q)(CH₂)_(p)—;    -   X^(E) is selected from a covalent bond, CR^(E2)R^(E3) and        NR^(E4);    -   R^(E2), R^(E3) and R^(E4) are independently selected from H and        C₁-C₆alkyl;    -   X^(H1)—X^(H2) is selected from —CR^(H2)R^(H3)—,        —CR^(H2)R^(H3)—CR^(H2)R^(H3)—, —C(O)CR^(H2)R^(H3)—,        —C(O)CR^(H2)R^(H3)—, —CR^(H2)R^(H3)C(O)—, —NR^(H4)C(O)—,        C(O)NR^(H4-), CR^(H2)R^(H3)S(O)₂ and —CR^(H2)═CR^(H2)—;    -   R^(H2), R^(H3) and R^(H4) are each independently selected from        H, C₁-C₆alkyl and P¹⁸;    -   X^(H3) is selected from N and CN;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, C₁-C₆alkenylene, arylene, heteroarylene,        C₁-C₆alkyleneoxy and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally        substituted with 1 to 4 substituents independently selected from        halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;    -   m is selected from 0 or 1;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6;    -   q is selected from 1, 2, 3 and 4; and    -   s is selected from 0 and 1.

Formula (G)—TLR8 Agonist

The TLR agonist can be a compound according to formula (G):

wherein:

-   -   P¹¹ is selected from H, C₁-C₆alkyl, C₁-C₆ alkoxy, NR^(V)R^(W)        and —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   P¹² is selected from H. C₁-C₆alkyl, aryl optionally substituted        by —C(O)NR^(V)R^(W), and —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   P¹³, P¹⁴ and P¹⁵ are independently selected from H, C₁-C₆alkyl,        C₁-C₆ alkoxy and —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   with the proviso that at least one of P¹¹, P¹², P¹³, P¹⁴ or P¹⁵        is —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   R^(V) and R^(W) are independently selected from H, C₁-C₆alkyl or        together with the nitrogen atom to which they are attached form        a 4 to 7 remembered heterocyclic ring;    -   X^(G) is selected from C, CH and N;    -   represents an optional double bond, wherein X^(G) is C if        is a double bond; and    -   R^(G) is selected from H and C₁-C₆alkyl;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and        —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1        to 4 substituents independently selected from halo, OH,        C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6 and    -   q is selected from 1, 2, 3 and 4.

Formulae (I) and (II)—TLR7 agonists [137]

The TLR agonist can be a compound according to formula (I) or formula(II):

wherein:

-   -   Z is —NH₂ or —OH;    -   X¹ is alkylene, substituted alkylene, alkenylene, substituted        alkenylene, alkynylene, substituted alkynylene, carbocyclylene,        substituted carbocyclylene, heterocyclylene, or substituted        heterocyclylene;    -   L¹ is a covalent bond, arylene, substituted arylene,        heterocyclylene, substituted heterocyclylene, carbocyclylene,        substituted carbocyclylene, —S—, —S(O)—, S(O)₂, —NR⁵—, or —O—    -   X² is a covalent bond, alkylene, or substituted alkylene;    -   L² is NR⁵—, —N(R⁵)C(O)—, —O—, —S—, —S(O)—, S(O)₂, or a covalent        bond;    -   R³ is H, alkyl, substituted alkyl, heteroalkyl, substituted        heteroalkyl, alkenyl, substituted alkenyl, aryl, substituted        aryl, arylalkyl, substituted arylalkyl, heterocyclyl,        substituted heterocyclyl, heterocyclylalkyl, or substituted        heterocyclylalkyl;    -   Y¹ and Y² are each independently a covalent bond, —O— or —NR⁵—;        or —Y¹—R¹ and —Y²—R² are each independently —O—N═C(R⁶R⁷);    -   R¹ and R² are each independently H, alkyl, substituted alkyl,        carbocyclyl, substituted carbocyclyl, heterocyclyl, substituted        heterocyclyl, alkenyl, substituted alkenyl, alkynyl, substituted        alkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl,        substituted heterocyclylalkyl, -alkylene-C(O)—O—R⁵,        -(substituted alkylene)-C(O)—O—R⁵, -alkylene-O—C(O)—R⁵,        -(substituted alkylene)-O—C(O)—R⁵, -alkylene-O—C(O)—O—R⁵, or        -(substituted alkylene)-O—C(O)—O—R⁵    -   R⁴ is H, halogen, —OH, —O-alkyl, —O-alkylene-O—C(O)—O—R⁵,        —O—C(O)—O—R⁵, —SH, or —NH(R⁵);    -   each R⁵, R⁶, and R⁷ are independently H, alkyl, substituted        alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl,        substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, arylalkyl, substituted arylalkyl,        heterocyclylalkyl, or substituted heterocyclylalkyl.

Formula (J) TLR2 Agonists [143]

The TLR agonist can be a compound according to formula (J):

wherein:

-   -   R¹ is H, —C(O)—C₇-C₁₈alkyl or —C(O)—C₁-C₆alkyl;    -   R² is C₇-C₁₈alkyl;    -   R³ is C₇-C₁₈alkyl;    -   L₁ is —CH₂OC(O)—, —CH₂O—, —CH₂NR⁷C(O)— or —CH₂OC(O)NR⁷—;    -   L₂ is —OC(O)—, —O—, —NR⁷C(O)— or —OC(O)NR⁷—;    -   R⁴ is -L₃R⁵ or -L₄R⁵;    -   R⁵ is —N(R⁷)₂, —OR⁷, —P(OR^(X))(OR⁷)₂, —C(O)OR⁷, —NR⁷C(O)L₃R⁸,        —NR⁷C(O)L₄R⁸, —OL₃R⁶, —C(O)NR⁷L₃R⁸, —C(O)NR⁷L₄R⁸. —S(O)₂OR⁷,        —OS(O)₂OR⁷, C₁-C₆alkyl, a C₆aryl, a C₁₀aryl, a C₁₄aryl, 5 to 14        ring membered heteroaryl containing 1 to 3 heteroatoms selected        from O, S and N, C₃-C₈cycloalkyl or a 5 to 6 ring membered        heterocycloalkyl containing 1 to 3 heteroatoms selected from O,        S and N, wherein the aryl, heteroaryl, cycloalkyl and        heterocycloalkyl of R⁵ are each unsubstituted or the aryl,        heteroaryl, cycloalkyl and heterocycloalkyl of R⁵ are cach        substituted with 1 to 3 substituents independently selected from        —OR⁹, —OL₃R⁶, —OL₄R⁶, —OR^(Y), and —C(O)OR;    -   L₃ is a C₁-C₁₀alkylene, wherein the C₁-C₁₀alkylene of L₃ is        unsubstituted, or the C₁-C₁₀alkylene of L₃ is substituted with 1        to 4 R⁶ groups, or the C₁-C₁₀alkylene of L₃ is substituted with        2 C₁-C₆alkyl groups on the same carbon atom which together,        along with the carbon atom they are attached to, form a        C₃-C₈cycloakyl;    -   L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)— or        —(CR¹¹R¹¹)((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)—, wherein each R¹¹ is        a C₁-C₆alkyl groups which together, along with the carbon atom        they are attached to, form a C₃-C₈cycloakyl;    -   each R⁶ is independently selected from halo, C₁-C₆alkyl,        C₁-C₆alkyl substituted with 1-2 hydroxyl groups, —OR⁷, —N(R⁷)₂,        —C(O)OH, —C(O)N(R⁷)₂, —P(OR^(X))(OR⁷)₂, a C₆aryl, a C₁₀aryl and        a C₁₄aryl;    -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   R⁸ is selected from —SR⁷, —C(O)OH, —P(O)(OR⁷)₂, and a 5 to 6        ring membered heterocycloalkyl containing 1 to 3 heteroatoms        selected from O and N;    -   R⁹ is phenyl;    -   each R¹⁰ is independently selected from H and halo;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6, and    -   q is 1, 2, 3 or 4.

Preferably R⁵ is P(O)(OR⁷)₂, —NR⁷C(O)L₃-P(O)(OR⁷)₂,—NR⁷C(O)L₄-P(OR^(X))(OR⁷)₂, —OL₃-P(O)(OR⁷)₂, —C(O)NR⁷L₃-P(O)(OR)₂, or—C(O)NR⁷L₄-P(O)(OR⁷)₂.

In some embodiments of (J), R₁ is H. In other embodiments of (J), R₁ is—C(O)—C₁₅alkyl;

In some embodiments of (J): (i) L₁ is —CH₂OC(O)— and L₂ is —OC(O)—, —O—,—NR⁷C(O)— or —OC(O)NR⁷—; or (ii) or L₁ is —CH₂O— and L₂ is —OC(O)—, —O—,—NR⁷C(O)— or —OC(O)NR⁷—; or (iii) L₁ is —CH₂NR⁷C(O)— and L₂ is —OC(O)—,—O—, —NR⁷C(O)— or —OC(O)NR⁷—; or (iv) L₁ is —CH₂OC(O)NR⁷— and L₂ is—OC(O)—, —O—, —NR⁷C(O)— or —OC(O)NR⁷—.

In some embodiments of (J): (i) L₁ is —CH₂OC(O)— and L₂ is —OC(O)—; or(ii) L₁ is —CH₂O— and L₂ is —O—; or (iii) L₁ is —CH₂O— and L₂ is—NHC(O)—; or (iv) L₁ is —CH₂OC(O)NH— and L₂ is —OC(O)NH—.

In some embodiments of (J), (i) R² is —C₁₁alkyl and R³ is —C₁₁alkyl; or(ii) R² is —C₁₆alkyl and R³ is —C₁₆alkyl; or (iii) R² is —C₁₆alkyl andR³ is —C₁₁alkyl; or (iv) R² is —C₁₂alkyl and R³ is —C₁₂alkyl; or (v) R²is —C₇alkyl and R³ is —C₇alkyl; or (vi) R² is —C₉alkyl and R³ is—C₉alkyl; or (vii) R² is —C₈alkyl and R³ is —C₈alkyl; or (viii) R² is—C₁₃alkyl and R³ is —C₁₃alkyl; or (ix) R² is —C₁₂alkyl and R³ is—C₁₁alkyl; or (x) R² is —C₁₂alkyl and R³ is —C₁₂alkyl; or (xi) R² is—C₁₀alkyl and R³ is —C₁₀alkyl; or (xii) R² is —C₁₈alkyl and R³ is—C₁₈alkyl.

In some embodiments of (J), R² is —C₁₁alkyl and R³ is —C₁₁alkyl.

In some embodiments of (J), L₃ is a C₁-C₁₀alkylene, wherein theC₁-C₁₀alkylene of L₃ is unsubstituted or is substituted with 1 to 4 R⁶groups.

In some embodiments of (J): L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)—;each R¹⁰ is independently selected from H and F; and each p isindependently selected from 2, 3, and 4.

In some embodiments of (J), each R⁶ is independently selected frommethyl, ethyl, i-propyl, i-butyl, —CH₂OH, —OH, —F, —NH₂, —C(O)OH,—C(O)NH₂, —P(O)(OH)₂ and phenyl.

In some embodiments of (J), each R⁷ is independently selected from H,methyl and ethyl.

TLR4 Agonists

Compositions of the invention can include a TLR4 agonist, and mostpreferably an agonist of human TLR4. TLR4 is expressed by cells of theinnate immune system, including conventional dendritic cells andmacrophages [144]. Triggering via TLR4 induces a signalling cascade thatutilizes both the MyD88- and TRIF-dependent pathways, leading to NF-κBand IRF3/7 activation, respectively. TLR4 activation typically inducesrobust IL-12p70 production and strongly enhances Th1-type cellular andhumoral immune responses.

Various useful TLR4 agonists are known in the art, many of which areanalogs of endotoxin or lipopolysaccharide (LPS). For instance, the TLR4agonist can be:

-   -   (i) 3d-MPL (i.e. 3-O-deacylated monophosphoryl lipid A; also        known as 3-de-O-acylated monophosphoryl lipid A or        3-O-desacyl-4′-monophosphoryl lipid A). This derivative of the        monophosphoryl lipid A portion of endotoxin has a de-acylated        position 3 of the reducing end of glucosamine. It has been        prepared from a heptoseless mutant of Salmonella minnesota, and        is chemically similar to lipid A but lacks an acid-labile        phosphoryl group and a base-labile acyl group. Preparation of        3d-MPL was originally described in ref. 145, and the product has        been manufactured and sold by Corixa Corporation. It is present        in GSK's ‘AS04’ adjuvant. Further details can be found in        references 146 to 149.    -   (ii) glucopyranosyl lipid A (GLA) [150] or its ammonium salt:

-   -   (iii) an aminoalkyl glucosaminide phosphate, such as RC-529 or        CRX-524 [151-153]. RC-529 and CRX-524 have the following        structure, differing by their R₂ groups:

-   -   (iv) compounds containing lipids linked to a        phosphate-containing acyclic backbone, such as the TLR4        antagonist E5564 [154,155]:

-   -   (v) A compound of formula I, II or III as defined in reference        156, or a salt thereof, such as compounds ‘ER 803058’, ‘ER        803732’, ‘ER 804053’, ‘ER 804058’, ‘ER 804059’, ‘ER 804442’, ‘ER        804680’, ‘ER 803022’, ‘ER 804764’ or ‘ER 804057’. ER 804057 is        also known as E6020 and it has the following structure:

-   -   -   whereas ER 803022 has the following structure:

-   -   (vi) One of the polypeptide ligands disclosed in reference 157.

Any of these TLR4 agonists can be used with the invention.

A composition of the invention can include an aluminium salt to whichthe TLR4 agonist is adsorbed. TLR4 agonists with adsorptive propertiestypically include a phosphorus-containing moiety which can undergoligand exchange with surface groups on an aluminium salt, andparticularly with a salt having surface hydroxide groups. Thus a usefulTLR4 agonist may include a phosphate, a phosphonate, a phosphinate, aphosphonite, a phosphinite, a phosphate, etc. Preferred TLR4 agonistsinclude at least one phosphate group [142] e.g. the agonists (i) to (v)listed above.

The preferred TLR4 agonist for use with the invention is 3d-MPL. Thiscan be adsorbed to an aluminium phosphate adjuvant, to an aluminiumhydroxide adjuvant, or to a mixture of both [158].

3d-MPL can take the form of a mixture of related molecules, varying bytheir acylation (e.g. having 3, 4, 5 or 6 acyl chains, which may be ofdifferent lengths). The two glucosamine (also known as2-deoxy-2-amino-glucose) monosaccharides are N-acylated at their2-position carbons (i.e. at positions 2 and 2′), and there is alsoO-acylation at the 3′ position. The group attached to carbon 2 hasformula —NH—CO—CH₂—CR¹R^(1′). The group attached to carbon 2′ hasformula —NH—CO—CH₂—CR²R^(2′). The group attached to carbon 3′ hasformula —O—CO—CH₂—CR³R^(3′). A representative structure is:

Groups R¹, R² and R³ are each independently —(CH₂)_(n)—CH₃. The value ofn is preferably between 8 and 16, more preferably between 9 and 12, andis most preferably 10.

Groups R^(1′), R^(2′) and R^(3′) can each independently be: (a) —H; (b)—OH; or (c) —O—CO—R⁴, where R⁴ is either —H or —(CH₂)_(m)—CH₃, whereinthe value of m is preferably between 8 and 16, and is more preferably10, 12 or 14. At the 2 position, in is preferably 14. At the 2′position, m is preferably 10. At the 3′ position, m is preferably 12.Groups R^(1′), R^(2′) and R^(3′) are thus preferably —O-acyl groups fromdodecanoic acid, tetradecanoic acid or hexadecanoic acid.

When all of R^(1′), R^(2′) and R^(3′) are —H then the 3d-MPL has only 3acyl chains (one on each of positions 2, 2′ and 3′). When only two ofR^(1′), R^(2′) and R^(3′) are —H then the 3d-MPL can have 4 acyl chains.When only one of R^(1′), R^(2′) and R^(3′) is —H then the 3d-MPL canhave 5 acyl chains. When none of R^(1′), R^(2′) and R^(3′) is —H thenthe 3d-MPL can have 6 acyl chains. The 3d-MPL used according to theinvention can be a mixture of these forms, with from 3 to 6 acyl chains,but it is preferred to include 3d-MPL with 6 acyl chains in the mixture,and in particular to ensure that the 6 acyl chain form makes up at least10% by weight of the total 3d-MPL e.g. ≧20%, ≧30%, ≧40%, ≧50% or more.3d-MPL with 6 acyl chains has been found to be the most adjuvant-activeform.

Thus the most preferred form of 3d-MPL for use with the invention is:

Where 3d-MPL is used in the form of a mixture then references to amountsor concentrations of 3d-MPL in compositions of the invention refer tothe combined 3d-MPL species in the mixture.

Typical compositions include 3d-MPL at a concentration of between 25μg/ml and 200 μg/ml e.g. in the range 50-150 μg/ml, 75-125 μg/ml, 90-110μg/ml, or about 100 μg/ml. It is usual to administer between 25-75 μg of3d-MPL per dose e.g. between 45-55 μg, or about 50 μg 3d-MPL per dose.

In aqueous conditions, 3d-MPL can form micellar aggregates or particleswith different sizes e.g. with a diameter <150 nm or >500 nm. Either orboth of these can be used with the invention, and the better particlescan be selected by routine assay. Smaller particles (e.g. small enoughto give a clear aqueous suspension of 3d-MPL) are preferred for useaccording to the invention because of their superior activity [159].Preferred particles have a mean diameter less than 150 nm, morepreferably less than 120 nm, and can even have a mean diameter less than100 nm. In most cases, however, the mean diameter will not be lower than50 nm. Where 3d-MPL is adsorbed to an aluminum salt then it may not bepossible to measure the 3D-MPL particle size directly, but particle sizecan be measured before adsorption takes place. Particle diameter can beassessed by the routine technique of dynamic light scattering, whichreveals a mean particle diameter. Where a particle is said to have adiameter of x nm, there will generally be a distribution of particlesabout this mean, but at least 50% by number (e.g. ≧60%, ≧70%, ≧80%,≧90%, or more) of the particles will have a diameter within the rangex±25%.

Formula (K) [160]

The TLR agonist can be a compound according to formula (K):

wherein:

-   -   R¹ is H, C₁-C₆alkyl, —C(R⁵)₂OH, -L¹R⁵, -L¹R⁶, -L²R⁵, -L²R⁶,        —OL²R⁵, or -OL²R⁶;    -   L¹ is —C(O)— or —O—;    -   L² is C₁-C₆alkylene, C₂-C₆alkenylene, arylene, heteroarylene or        —((CR⁴R⁴)_(p)O)_(q)(CH₂)_(p), wherein the C₁-C₆alkylene and        C₂-C₆alkenylene of L² are optionally substituted with 1 to 4        fluoro groups;    -   each L³ is independently selected from C₁-C₆alkylene and        —((CR⁴R⁴)_(p)O)_(q)(CH₂)_(p)—, wherein the C₁-C₆alkylene of L³        is optionally substituted with 1 to 4 fluoro groups;    -   L⁴ is arylene or heteroarylene;    -   R² is H or C₁-C₆alkyl;    -   R³ is selected from C₁-C₄alkyl, -L³R⁵, -L¹R⁵, -L³R⁷, -L³L⁴L³R⁷,        -L³L⁴R⁵, -L³L⁴L³R⁵, —OL³R⁷, —OL³R⁷, —OL³L⁴R⁷, —OL³L⁴L³R⁷, —OR,        —OL³L⁴R⁵, —OL³L⁴L³R⁵ and —C(R⁵)₂OH;    -   each R⁴ is independently selected from H and fluoro;    -   R⁵ is —P(O)(OR⁹)₂,    -   R⁶ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰;    -   R⁷ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰;    -   R⁸ is H or C₁-C₄alkyl;    -   each R⁹ is independently selected from H and C₁-C₆alkyl;    -   R¹⁰ is H or C₁-C₄alkyl;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6, and    -   q is 1, 2, 3 or 4.

The compound of formula (K) is preferably of formula (K′):

wherein:

-   -   P¹ is selected from H, C₁-C₆alkyl optionally substituted with        COOH and —Y-L-X—P(O)(OR^(X))(OR^(Y)); P² is selected from H,        C₁-C₆alkyl, C₁-C₆alkoxy and —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   with the proviso that at least one of P¹ and P² is        —Y-L-X—P(OR^(X))(OR^(X)X)(OR^(Y));    -   R^(B) is selected from H and C₁-C₆alkyl;    -   R^(X) and R^(Y) are independently selected from H and        C₁-C₆alkyl;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and        —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1        to 4 substituents independently selected from halo, OH,        C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6; and    -   q is selected from 1, 2, 3 and 4.

In some embodiments of formula (K′): P¹ is selected from C₁-C₆alkyloptionally substituted with COOH and —Y-L-X—P(O)(OR^(X))(OR^(Y)); P² isselected from C₁-C₆alkoxy and —Y-L-X—P(O)(OR^(X))(OR^(Y)); R^(B) isC₁-C₆alkyl; X is a covalent bond; L is selected from C₁-C₆alkylene and—((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4substituents independently selected from halo, OH, C₁-C₄alkyl,—OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2and 3; q is selected from 1 and 2.

A preferred TLR7 agonist of formula K is3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[f]-[1,7]naphthyridin-8-yl)propanoic acid, referred to herein ascompound “K2”:

The K2 compound can also be used as an arginine salt monohydrate.

Formula (F)—TLR7 Agonists [138]

The TLR agonist can be a compound according to formula (F):

-   -   wherein:        -   X³ is N;        -   X⁴ is N or CR³        -   X⁵ is —CR₄═CR₅—;        -   R¹ and R² are H;        -   R³ is H;        -   R⁴ and R are each independently selected from H, halogen,            —C(O)OR⁷, —C(O)R⁷, —C(O)N(R¹¹R¹²), —N(R¹¹R¹²), —N(R⁹)₂,            —NHN(R⁹)₂, —SR⁷, —(CH₂)_(n)OR⁷, —(CH₂)_(n)R⁷, - LR⁸, -LR¹⁰,            —OLR⁸, —OLR¹⁰, C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,            C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy,            aryl, heteroaryl, C₃-C₈cycloalkyl, and            C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl,            C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne,            C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl,            C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R⁴ and            R⁵ are each optionally substituted with 1 to 3 substituents            independently selected from halogen, —CN, —NO₂, —R⁷, —OR⁸,            —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂,            —OP(O)(OR⁸)₂, —P(O)(0R¹⁰)₂. —OP(OR)(OR¹⁰)₂, —C(O)N(R⁹)₂,            —S(O)₂R⁸, —S(O)R⁸, —S(O)₂N(R)₂, and —NR⁹S(O)₂R⁸;    -   or, R¹ and R⁴, or R⁴ and R⁵, or R⁵ and R⁶, when present on        adjacent ring atoms, can optionally be linked together to form a        5-6 membered ring, wherein the 5-6 membered ring is optionally        substituted with R⁷;    -   each L is independently selected from a bond,        —(O(CH₂)_(m))_(t)—, C₁-C₆alkyl, C₂-C₆alkenylene and        C₂-C₆alkynylene, wherein the C₁-C₆alkyl, C₂-C₆alkenylene and        C₂-C₆alkynylene of L are each optionally substituted with 1 to 4        substituents independently selected from halogen, —R⁸, —OR⁸,        —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, and        —OP(O)(OR¹⁰)₂;    -   R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl,        C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,        C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and        C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl, heteroaryl,        C₃-C₈cycloalkyl, C₁-C₆heteroalkyl. C₁-C₆haloalkyl, C₂-C₈alkene,        C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and        C₃-C₈heterocycloalkyl groups of R⁷ are each optionally        substituted with 1 to 3 R¹³ groups, and each R¹³ is        independently selected from halogen, —CN, -LR⁹, -LOR⁹, —OLR⁹,        -LR¹¹, -LOR¹⁰, —OLR¹⁰, -LR⁸, -LOR⁸, —OLR⁸, -LSR⁸, -LSR¹⁰,        -LC(O)R⁸, —OLC(O)R⁸, -LC(O)OR⁸, -LC(O)R¹⁰, -LOC(O)OR⁸,        -LC(O)NR⁹R¹¹, -LC(O)NR⁹R⁸, -LN(R⁹)₂, -LNR⁹R⁸, -LNR⁹R¹⁰,        -LC(O)N(R⁹)₂, -LS(O)₂R⁸, -LS(O)R⁸, -LC(O)NR⁸OH, -LNR⁹C(O)R⁸,        -LNR⁹C(O)OR⁸, -LS(O)₂N(R⁹)₂, —OLS(O)₂N(R⁹)₂, -LNR⁹S(O)₂R⁸,        -LC(O)NR⁹LN(R⁹)₂, -LP(O)(OR⁹)₂, -LOP(O)(OR⁸)₂, -LP(O)(OR¹⁰)₂ and        -OLP(O)(OR¹⁰)₂;    -   each R⁸ is independently selected from H, —CH(R¹⁰)₂, C₁-C₈alkyl,        C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl, C₁-C₆alkoxy,        C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl,        C₂-C₆hydroxyalkyl and C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl,        C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,        C₁-C₆alkoxy, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl,        C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy groups of R⁸ are each        optionally substituted with 1 to 3 substituents independently        selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹,        —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹⁰, —NR¹¹R¹², —N(R⁹)₂,        —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹, —S(O)R¹¹,        —S(O)₂NR¹¹R¹¹, —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂;    -   each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸,        —C(O)R¹⁰, —C(O)OR¹⁰, —S(O)₂R¹⁰, —C₁-C₆ alkyl, C₁-C₆ heteroalkyl        and C₃-C₆ cycloalkyl, or each R⁹ is independently a C₁-C₆alkyl        that together with N they are attached to form a        C₃-C₈heterocycloalkyl, wherein the C₃-C₈heterocycloalkyl ring        optionally contains an additional heteroatom selected from N, O        and S, and wherein the C₁-C₆ alkyl, C₁-C₆ heteroalkyl, C₃-C₆        cycloalkyl, or C₃-C₈heterocycloalkyl groups of R⁹ are each        optionally substituted with 1 to 3 substituents independently        selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, OC(O)R¹¹,        —C(O)0R¹¹, —NR¹¹R¹², —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹,        —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹) and        —OP(O)(OR¹¹)₂;    -   each R¹⁰ is independently selected from aryl, C₃-C₈cycloalkyl,        C₃-C₈heterocycloalkyl and heteroaryl, wherein the aryl,        C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl groups are        optionally substituted with 1 to 3 substituents selected from        halogen, —R⁸, —OR⁸, -LR⁹, -LOR⁹, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹CO₂R⁸.        —CO₂R⁸, —C(O)R and —C(O)N(R⁹)₂,    -   R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl,        C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl,        C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the        C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl,        C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R¹¹ and R¹²        are each optionally substituted with 1 to 3 substituents        independently selected from halogen, —CN, R⁸, —OR⁸, C(O)R⁸,        OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —NR⁸C(O)R⁸, —NR⁸C(O)OR⁸,        —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl, —S(O)₂R⁸, —S(O)₂N(R⁹)₂,        —NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and C₁-C₆haloalkoxy;    -   or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken        together with the N atom to which they are attached form an        optionally substituted C₃-C₈heterocycloalkyl ring optionally        containing an additional heteroatom selected from N, O and S;    -   ring A is an aryl or a heteroaryl, wherein the aryl and        heteroaryl groups of Ring A are optionally substituted with 1 to        3 R^(A) groups, wherein each R^(A) is independently selected        from —R⁸, —R⁷, —OR⁷, —OR⁸, —R¹⁰, —OR¹⁰, —SR⁸, —NO₂, —CN,        —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹C(S)R⁸, —NR⁹C(O)N(R⁹)₂, —NR⁹C(S)N(R⁹)₂,        —NR⁹CO₂R⁸, —NR⁹NR⁹C(O)R⁸, —NR⁹NR⁹C(O)N(R⁹)₂, —NR⁹NR⁹CO₂R⁸,        —C(O)C(O)R⁸, —C(O)CH₂C(O)R⁸, —CO₂R⁸, —(CH₂)_(n)CO₂R⁸, —C(O)R⁸,        —C(S)R⁸, —C(O)N(R⁹)₂, —C(S)N(R⁹)₂, —OC(O)N(R⁹)₂, —OC(O)R⁸,        —C(O)N(OR¹¹)R⁸, —C(NOR)R⁸, —S(O)₂R⁸, —S(O)₃R⁸, —SO₂N(R⁹)₂,        —S(O)R⁸, —NR⁹SO₂N(R⁹)₂, —NR⁹SO₂R⁸, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂,        —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —N(0R⁸)R⁸, —CH═CHCO₂R⁸,        —C(═NH)—N(R⁹)₂, and —(CH₂)_(n)NHC(O)R⁸ or two adjacent R^(A)        substituents on Ring A form a 5-6 membered ring that contains up        to two heteroatoms as ring members;    -   n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6, 7        or 8;    -   each m is independently selected from 1, 2, 3, 4, 5 and 6, and    -   t is 1, 2, 3, 4, 5, 6, 7 or 8.

Formulae (C), (D), (E), (G) and (H)

As discussed above, the TLR agonist can be of formula (C), (D), (E), (G)or (H).

The ‘parent’ compounds of formulae (C), (D), (E) and (H) are useful TLR7agonists (see references 136-139 and 161-177) but are preferablymodified herein by attachment of a phosphorus-containing moiety.

In some embodiments of formulae (C), (D) and (E) the compounds havestructures according to formulae (C′), (D′) and (E′), shown below:

The embodiments of the invention of formulae (C), (D), (E) and (H) alsoapply to formulae (C′), (D′), (E′) and (H′).

In some embodiments of formulae (C), (D), (E), and (H): X is O; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (C): P³ is selected from C₁-C₆alkyl,CF₃, and —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and—Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁴ is selected from —C₁-C₆alkylaryl and—Y-L-X—P(O)(OR^(X))(OR^(Y)); X^(C) is CH; X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p isindependently selected from 1, 2 and 3: q is 1 or 2.

In other embodiments of formulae (C), (D), (E), and (H): X is a covalentbond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)—each optionally substituted with 1 to 4 substituents independentlyselected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each pis independently selected from 1, 2 and 3; and q is selected from 1 and2.

In other embodiments of formula (C): P³ is selected from C₁-C₆alkyl,CF₃, and —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and—Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁴ is selected from —C₁-C₆alkylaryl and—Y-L-X—P(O)(OR^(X))(OR^(Y)); X^(C) is N; X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; q is selected from 1 and 2.

In other embodiments of formula (D): P⁵ is selected from C₁-C₆alkyl, and—Y-L-X—P(O)(OR^(X))(OR^(Y)).

In other embodiments of formula (D): X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (D): X is a covalent bond; L is selectedfrom C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X is a covalent bond; L is selectedfrom C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X^(E) is CH₂, P⁸ is C₁-C₆alkoxyoptionally substituted with —Y-L-X—P(O)(OR^(X))(OR^(Y)).

In other embodiments of formula (E): P⁹ is —NHC₁-C₆alkyl optionallysubstituted with OH and C₁-C₆alkyl, and —Y-L-X—P(O)(OR^(X))(OR^(Y)).

In some embodiments, a compound of formula (C) is not a compound inwhich P⁴ is —Y-L-X—P(O)(OR^(X))(OR^(Y)).

In some embodiments, in a compound of formula (C), P⁴ is selected fromH, C₁-C₆alkyl, —C₁-C₆alkylaryl.

In some embodiments of formula (H): X^(H)—X^(H2) is CR^(H2)R^(H3),R^(H2) and R^(H3) are H, X^(H3) is N, X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

In some embodiments of formula (H): X^(H1)-X^(H2) is CR^(H2)R^(H3),R^(H2) and R^(H3) are H. X^(H3) is N, X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

The ‘parent’ compounds of formula (G) are useful TLR8 agonists (seereferences 140 & 141) but are preferably modified herein by attachmentof a phosphorus-containing moiety to permit adsorption. In someembodiments of formula (G), the compounds have structures according toformula (G′);

In some embodiments of formula (G) or (G′): X^(G) is C and

represents a double bond. In some embodiments of formula (G) or (G′): Xis a covalent bond; L is selected from C₁-C₆alkylene and—((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4substituents independently selected from halo, OH, C₁-C₄alkyl,—OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2and 3; and q is selected from 1 and 2.

In some embodiments of formula (G) or (G′): X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

Immunogenic Compositions

In addition to the antigen and adjuvant components discussed above,compositions of the invention may comprise further non-antigeniccomponent(s). These can include carriers, excipients, buffers, etc.These non-antigenic components may have various sources. For example,they may be present in one of the antigen or adjuvant materials that isused during manufacture or may be added separately from thosecomponents.

Preferred compositions of the invention include one or morepharmaceutical carrier(s) and/or excipient(s).

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml.

Compositions will generally have an osmolality of between 200 mOsm/kgand 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 280-320 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination [178], but keeping osmolality in this range is neverthelesspreferred.

Compositions of the invention may include one or more buffers. Typicalbuffers include: a phosphate buffer; a Tris buffer; a borate buffer; asuccinate buffer; a histidine buffer; or a citrate buffer. Buffers willtypically be included in the 5-20 mM range.

The pH of a composition of the invention will generally be between 6.0and 7.5. A manufacturing process may therefore include a step ofadjusting the pH of a composition prior to packaging. Aqueouscompositions administered to a patient can have a pH of between 5.0 and7.5, and more typically between 5.0 and 6.0 for optimum stability; wherea diphtheria toxoid and/or tetanus toxoid is present, the pH is ideallybetween 6.0 and 7.0.

Compositions of the invention are preferably sterile.

Compositions of the invention are preferably non-pyrogenic e.g.containing <1 EU (endotoxin unit, a standard measure; 1 EU is equal to0.2 ng FDA reference standard Endotoxin EC-2 ‘RSE’) per dose, andpreferably <0.1 EU per dose.

Compositions of the invention are preferably gluten free.

If a composition includes adsorbed component then it may be a suspensionwith a cloudy appearance. This appearance means that microbialcontamination is not readily visible, and so the vaccine preferablycontains an antimicrobial agent. This is particularly important when thevaccine is packaged in multidose containers. Preferred antimicrobialsfor inclusion are 2-phenoxyethanol and thimerosal. It is preferred,however, not to use mercurial preservatives (e.g. thimerosal) during theprocess of the invention. Thus, between 1 and all of the componentsmixed in a process may be substantially free from mercurialpreservative. However, the presence of trace amounts may be unavoidableif a component was treated with such a preservative before being used inthe invention. For safety, however, it is preferred that the finalcomposition contains less than about 25 ng/ml mercury. More preferably,the final vaccine product contains no detectable thimerosal. This willgenerally be achieved by removing the mercurial preservative from anantigen preparation prior to its addition in the process of theinvention or by avoiding the use of thimerosal during the preparation ofthe components used to make the composition. Mercury-free compositionsare preferred.

Compositions of the invention will usually be in aqueous form.

During manufacture, dilution of components to give desired finalconcentrations will usually be performed with WFI (water for injection),or with buffer.

The invention can provide bulk material which is suitable for packaginginto individual doses, which can then be distributed for administrationto patients. Concentrations discussed above are typically concentrationsin final packaged dose, and so concentrations in bulk vaccine may behigher (e.g. to be reduced to final concentrations by dilution).

Compositions of the invention are administered to patients in unit dosesi.e. the amount of a composition given to a single patient in a singleadministration (e.g. a single injection is a unit dose). Where acomposition is administered as a liquid then a unit dose typically has avolume of 0.5 ml. This volume will be understood to include normalvariance e.g. 0.5 ml±0.05 ml. For multidose situations, multiple doseamounts will be extracted and packaged together in a single containere.g. 5 ml for a 10-dose multidose container (or 5.5 ml with 10%overfill).

Residual material from individual antigenic components may also bepresent in trace amounts in the final vaccine produced by the process ofthe invention. For example, if formaldehyde is used to prepare thetoxoids of diphtheria, tetanus and pertussis then the final vaccineproduct may retain trace amounts of formaldehyde (e.g. less than 10μg/ml, preferably <5 μg/ml). Media or stabilizers may have been usedduring poliovirus preparation (e.g. Medium 199), and these may carrythrough to the final vaccine. Similarly, free amino acids (e.g. alanine,arginine, aspartate, cysteine and/or cystine, glutamate, glutamine,glycine, histidine, proline and/or hydroxyproline, isoleucine, leucine,lysine, methionine, phenylalanine, serine, threonine, tryptophan,tyrosine and/or valine), vitamins (e.g. choline, ascorbate, etc.),disodium phosphate, monopotassium phosphate, calcium, glucose, adeninesulfate, phenol red, sodium acetate, potassium chloride, etc. may beretained in the final vaccine at ≦100 μg/ml, preferably <10 μg/ml, each.Other components from antigen preparations, such as neomycin (e.g.neomycin sulfate, particularly from a poliovirus component), polymyxin B(e.g. polymyxin B sulfate, particularly from a poliovirus component),etc. may also be present at sub-nanogram amounts per dose. A furtherpossible component of the final vaccine which originates in the antigenpreparations arises from less-than-total purification of antigens. Smallamounts of B. pertussis. C. diphtheriae. C. tetani and S. cerevisiaeproteins and/or genomic DNA may therefore be present. To minimize theamounts of these residual components, antigen preparations arepreferably treated to remove them prior to the antigens being used withthe invention.

Where a poliovirus component is used, it will generally have been grownon Vero cells. The final vaccine preferably contains less than 10 ng/ml,preferably ≦1 ng/ml e.g. ≦500 μg/ml or ≦50 μg/ml of Vero cell DNA e.g.less than 10 ng/ml of Vero cell DNA that is ≧50 base pairs long.

Compositions of the invention are presented for use in containers.Suitable containers include vials and disposable syringes (preferablysterile ones). Processes of the invention may comprise a step ofpackaging the vaccine into containers for use. Suitable containersinclude vials and disposable syringes (preferably sterile ones).

The invention also provides a delivery device (e.g. syringe, nebuliser,sprayer, inhaler, dermal patch, etc.) containing a pharmaceuticalcomposition of the invention e.g. containing a unit dose.

This device can be used to administer the composition to a vertebratesubject.

The invention also provides a sterile container (e.g. a vial) containinga pharmaceutical composition of the invention e.g. containing a unitdose.

The invention also provides a unit dose of a pharmaceutical compositionof the invention.

The invention also provides a hermetically scaled container containing apharmaceutical composition of the invention. Suitable containers includee.g. a vial.

Where a composition of the invention is presented in a vial, this ispreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex-sensitive patients, vials may be sealed with a latex-free stopper.The vial may include a single dose of vaccine, or it may include morethan one dose (a ‘multidose’ vial) e.g. 10 doses. When using a multidosevial, each dose should be withdrawn with a sterile needle and syringeunder strict aseptic conditions, taking care to avoid contaminating thevial contents.

Preferred vials are made of colorless glass.

A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial (e.g. to reconstitute lyophilised materialtherein), and the contents of the vial can be removed back into thesyringe. After removal of the syringe from the vial, a needle can thenbe attached and the composition can be administered to a patient. Thecap is preferably located inside a seal or cover, such that the seal orcover has to be removed before the cap can be accessed.

Where the composition is packaged into a syringe, the syringe will notnormally have a needle attached to it, although a separate needle may besupplied with the syringe for assembly and use.

Safety needles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and⅝-inch 25-gauge needles are typical. Syringes may be provided withpeel-off labels on which the lot number and expiration date of thecontents may be printed, to facilitate record keeping. The plunger inthe syringe preferably has a stopper to prevent the plunger from beingaccidentally removed during aspiration. The syringes may have a latexrubber cap and/or plunger. Disposable syringes contain a single dose ofvaccine. The syringe will generally have a tip cap to seal the tip priorto attachment of a needle, and the tip cap is preferably made of butylrubber. If the syringe and needle are packaged separately then theneedle is preferably fitted with a butyl rubber shield. Grey butylrubber is preferred. Preferred syringes are those marketed under thetrade name “Tip-Lok”™.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

After a composition is packaged into a container, the container can thenbe enclosed within a box for distribution e.g. inside a cardboard box,and the box will be labeled with details of the vaccine e.g. its tradename, a list of the antigens in the vaccine (e.g. ‘hepatitis Brecombinant’, etc.), the presentation container (e.g. ‘DisposablePrefilled Tip-Lok Syringes’ or ‘10×0.5 ml Single-Dose Vials’), its dose(e.g. ‘each containing one 0.5 ml dose’), warnings (e.g. ‘For Adult UseOnly’ or ‘For Pediatric Use Only’), an expiration date, an indication, apatent number, etc. Each box might contain more than one packagedvaccine e.g. five or ten packaged vaccines (particularly for vials).

The vaccine may be packaged together (e.g. in the same box) with aleaflet including details of the vaccine e.g. instructions foradministration, details of the antigens within the vaccine, etc. Theinstructions may also contain warnings e.g. to keep a solution ofadrenaline readily available in case of anaphylactic reaction followingvaccination, etc.

The packaged vaccine is preferably stored at between 2° C. and 8° C. Itshould not be frozen.

Vaccines can be provided in full-liquid form (i.e. where all antigeniccomponents are in aqueous solution or suspension) after manufacture, orthey can be prepared in a form where the vaccine can be preparedextemporaneously at the time/point of use by mixing together twocomponents. Such two-component embodiments include liquid/liquid mixingand liquid/solid mixing e.g. by mixing aqueous material with lyophilisedmaterial. For instance, in one embodiment a vaccine can be made bymixing: (a) a first component comprising aqueous antigens and/oradjuvant; and (b) a second component comprising lyophilized antigens. Inanother embodiment a vaccine can be made by mixing: (a) a firstcomponent comprising aqueous antigens and/or adjuvant; and (b) a secondcomponent comprising aqueous antigens. In another embodiment a vaccinecan be made by mixing: (a) a first component comprising aqueousantigens; and (b) a second component comprising aqueous adjuvant. Thetwo components are preferably in separate containers (e.g. vials and/orsyringes), and the invention provides a kit comprising components (a)and (b).

Another useful liquid/lyophilised format comprises (a) an aqueouscomplex of an aluminium salt and a TLR agonist and (b) a lyophilisedcomponent including one or more antigens. A vaccine composition suitablefor patient administration is obtained by mixing components (a) and (b).In some embodiments component (a) is antigen-free, such that allantigenic components in the final vaccine are derived from component(b); in other embodiments component (a) includes one or more antigen(s),such that the antigenic components in the final vaccine are derived fromboth components (a) and (b).

Thus the invention provides a kit for preparing a combination vaccine,comprising components (a) and (b) as noted above. The kit components aretypically vials or syringes, and a single kit may contain both a vialand a syringe. The invention also provides a process for preparing sucha kit, comprising the following steps: (i) preparing an aqueouscomponent vaccine as described above; (ii) packaging said aqueouscombination vaccine in a first container e.g. a syringe; (iii) preparingan antigen-containing component in lyophilised form; (iv) packaging saidlyophilised antigen in a second container e.g. a vial; and (v) packagingthe first container and second container together in a kit. The kit canthen be distributed to physicians.

A liquid/lyophilised format is particularly useful for vaccines thatinclude a conjugate component, particularly Hib and/or meningococcaland/or pneumococcal conjugates, as these may be more stable inlyophilized form. Thus conjugates may be lyophilised prior to their usewith the invention.

Where a component is lyophilised it generally includes non-activecomponents which were added prior to freeze-drying e.g. as stabilizers.Preferred stabilizers for inclusion are lactose, sucrose and mannitol,as well as mixtures thereof e.g. lactose/sucrose mixtures,sucrose/mannitol mixtures, etc. A final vaccine obtained by aqueousreconstitution of the lyophilised material may thus contain lactoseand/or sucrose. It is preferred to use amorphous excipients and/oramorphous buffers when preparing lyophilised vaccines [179].

Most compositions of the invention include diphtheria, tetanus andpertussis toxoids. In pediatric-type compositions the compositionincludes an excess of diphtheria toxoid relative to tetanus toxoid (asmeasured in Lf units). The excess is ideally at least 1.5:1 e.g. 5 Lf ofdiphtheria toxoid for every 2 Lf of tetanus toxoid (i.e. a 2.5:1 ratio).These embodiments are most useful in infants and children. Inbooster-type compositions, which are most useful in adolescents andadults, the composition includes an excess of tetanus toxoid relative todiphtheria toxoid (as measured in Lf units). The excess is ideally atleast 1.5:1 e.g. 2 Lf of tetanus toxoid for every 1 Lf of diphtheriatoxoid (i.e. a 2:1 ratio). In further embodiments, equal amounts ofdiphtheria and tetanus toxoids are used (in Lf units). Where one ofdiphtheria or tetanus is present at an excess, the excess should ideallybe at least 1.5-fold e.g. 2-fold or 2.5-fold, but the excess will notusually be more than 5-fold.

A composition of the invention includes a serogroup B meningococcusimmunogen and at least one of a diphtheria toxoid, a tetanus toxoid,and/or a pertussis toxoid. Ideally a composition includes all four of aserogroup B meningococcus immunogen, a diphtheria toxoid, a tetanustoxoid, and a pertussis toxoid. In some embodiments a composition of theinvention includes no immunogens beyond those in this list; in otherembodiments a composition of the invention does include immunogensbeyond those in this list. Thus, for example, some compositions includediphtheria, tetanus and pertussis toxoids, inactivated poliovirus forTypes 1, 2 & 3, hepatitis B virus surface antigen and a Hib conjugate.The antigenic portion of these compositions may consist of the antigensin this list, or may further include antigens from additional pathogens(e.g. meningococcus). Thus the compositions can be used as vaccinesthemselves, or as components of further combination vaccines.

Specific embodiments of the invention include compositions whoseimmunogens consist of: (a) D-T-aP-MenB; (b) D-T-aP-MenB-IPV; (c)D-T-aP-MenB-HBsAg; (d) D-T-aP-MenB-Hib; (e) D-T-aP-MenB-HBsAg-Hib; (f)D-T-aP-MenB-HBsAg-IPV; (g) D-T-aP-MenB-IPV-Hib; (h)D-T-aP-MenB-IPV-Hib-HBsAg; (i) D-T-MenB; where “D” is diphtheria toxoid,‘T’ is tetanus toxoid, “aP” is an acellular pertussis antigen ormixture, MenB is a serogroup B meningococcus antigen or mixture, “IPV”is an inactivated poliovirus antigen or mixture, “HBsAg” is a hepatitisB virus surface antigen, and “Hib” is a conjugated H. influenzae type Bcapsular saccharide.

Methods of Treatment, and Administration of the Vaccine

Compositions of the invention are suitable for administration to humanpatients, and the invention provides a method of raising an immuneresponse in a patient, comprising the step of administering acomposition of the invention to the patient.

The invention also provides a composition of the invention for use inmedicine. The composition may be administered as variously describedherein e.g. in some embodiments by giving an infant no more than twodoses of a combination vaccine.

The invention also provides the use of a serogroup B meningococcusimmunogen, a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid(and, optionally, an adjuvant) in the manufacture of a medicament forraising an immune response in a patient. The medicament is ideally acomposition as variously described elsewhere herein, and it can beadministered as variously described herein.

The immune responses raised by these methods, uses and compositions areideally protective, and immunogenic compositions of the invention arepreferably vaccines, for use in the prevention of at least diphtheria,tetanus, and whooping cough. Depending on their antigen components thevaccines may also protect against bacterial meningitis, polio,hepatitis, etc.

In order to have full efficacy, a typical primary immunization schedule(particularly for a child) may involve administering more than one dose.For example, doses may be at: 0 & 6 months (time 0 being the firstdose); at 0, 1, 2 & 6 months; at day 0, day 21 and then a third dosebetween 6 & 12 months; at 2, 4 & 6 months; at 3, 4 & 5 months; at 6, 10& 14 weeks; at 2, 3 & 4 months; or at 0, 1, 2, 6 & 12 months.

Compositions can also be used as booster doses e.g. for children in thesecond year of life, for an adolescent, or for an adult.

Compositions of the invention can be administered by intramuscularinjection e.g. into the arm or leg.

Optional Requirements and Disclaimers [180]

In some embodiments, the invention does not encompass compositions inunit dose form comprising (i) a diphtheria toxoid, a tetanus toxoid, anda pertussis toxoid, and (ii) an aluminium salt adjuvant, wherein theamount of Al⁺⁺⁺ in the unit dose is less than 0.2 mg. In otherembodiments, if a composition is in unit dose form and comprises (i) adiphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, and (ii) analuminium salt adjuvant, but the amount of Al⁺⁺⁺ in the unit dose isless than 0.2 mg, then: (a) the composition includes at least a 1.5-foldexcess of diphtheria toxoid to tetanus toxoid, measured in Lf units; or(b) the composition includes at least a 1.5-fold excess of tetanustoxoid to diphtheria toxoid, measured in Lf units; or (c) thecomposition includes an acellular PT-containing antigen pertussisantigen rather than a whole-cell pertussis antigen.

In some embodiments, the invention does not encompass compositionscomprising (i) a diphtheria toxoid, a tetanus toxoid, and a pertussistoxoid, and (ii) an aluminium salt adjuvant, wherein the concentrationof Al⁺⁺⁺ is less than 0.4 mg/ml. In other embodiments, if a compositioncomprises (i) a diphtheria toxoid, a tetanus toxoid, and a pertussistoxoid, and (ii) an aluminium salt adjuvant, but the concentration ofAl⁺⁺⁺ in the unit dose is less than 0.4 mg/ml, then: (a) the compositionincludes at least a 1.5-fold excess of diphtheria toxoid to tetanustoxoid, measured in Lf units; or (b) the composition includes at least a1.5-fold excess of tetanus toxoid to diphtheria toxoid, measured in Lfunits; or (c) the composition includes an acellular PT-containingantigen pertussis antigen rather than a whole-cell pertussis antigen.

In some embodiments, the invention does not encompass compositionscomprising (i) an aluminium salt adjuvant and (ii) ≦8 Lf/ml diphtheriatoxoid, ≦3.5 Lf/ml tetanus toxoid, and ≦5 μg/ml pertussis toxoid. Inother embodiments, if a composition comprises (i) an aluminium saltadjuvant and (ii) ≦8 Lf/ml diphtheria toxoid, ≦3.5 Lf/ml tetanus toxoid,and ≦5 μg/ml pertussis toxoid, then: (a) the composition includes atleast a 1.5-fold excess of diphtheria toxoid to tetanus toxoid, measuredin Lf units; or (b) the composition includes at least a 1.5-fold excessof tetanus toxoid to diphtheria toxoid, measured in Lf units; or (c) thecomposition includes an acellular PT-containing antigen pertussisantigen rather than a whole-cell pertussis antigen.

In some embodiments, the invention does not encompass compositionscomprising (i) an oil-in-water emulsion adjuvant (ii) a diphtheriatoxoid, a tetanus toxoid, a pertussis toxoid, and a Hib conjugate, and(iii) a hepatitis B virus surface antigen and/or an inactivatedpoliovirus antigen. In other embodiments, if a composition comprises (i)an oil-in-water emulsion adjuvant (ii) a diphtheria toxoid, a tetanustoxoid, a pertussis toxoid, and a Hib conjugate, then: (a) thecomposition does not include a hepatitis B virus surface antigen; or (b)the composition does not include an inactivated poliovirus antigen; or(c) the composition includes neither an inactivated poliovirus antigennor a hepatitis B virus surface antigen; or (d) the composition includesat least a 1.5-fold excess of diphtheria toxoid to tetanus toxoid,measured in Lf units; or (e) the composition includes at least a1.5-fold excess of tetanus toxoid to diphtheria toxoid, measured in Lfunits; or (f) the composition includes an acellular PT-containingantigen pertussis antigen rather than a whole-cell pertussis antigen.

In some embodiments, the invention does not encompass compositions whichcomprise a conjugate of a H. influenzae type b capsular saccharideantigen and an outer membrane protein complex from serogroup Bmeningococcus. In other embodiments, if a composition of the inventionincludes a conjugate of a H. influenzae type b capsular saccharideantigen and an outer membrane protein complex from serogroup Bmeningococcus then it must also include a further immunogen fromserogroup B meningococcus.

In some embodiments, the invention does not encompass compositions whichinclude both an aluminium salt adjuvant and a TLR4 agonist.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means, for example,x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where an antigen is described as being “adsorbed” to an adjuvant, it ispreferred that at least 50% (by weight) of that antigen is adsorbed e.g.50%, 60%, 70%, 80%, 90%, 95%, 98% or more. It is preferred thatdiphtheria toxoid and tetanus toxoid are both totally adsorbed i.e. noneis detectable in supernatant. Total adsorption of HBsAg can be used.

Amounts of conjugates are generally given in terms of mass of saccharide(i.e. the dose of the conjugate (carrier+saccharide) as a whole ishigher than the stated dose) in order to avoid variation due to choiceof carrier.

Phosphorous-containing groups employed with the invention may exist in anumber of protonated and deprotonated forms depending on the pH of thesurrounding environment, for example the pH of the solvent in which theyare dissolved. Therefore, although a particular form may be illustratedherein, it is intended, unless otherwise mentioned, for theseillustrations to merely be representative and not limiting to a specificprotonated or deprotonated form. For example, in the case of a phosphategroup, this has been illustrated as —OP(OX)(OH)₂ but the definitionincludes the protonated forms —[OP(O)(OH₂)(OH)]⁺ and [OP(O)(OH₂)₂]²⁺that may exist in acidic conditions and the deprotonated forms—[OP(O)(OH)(O)] and [OP(OXO)₂]²⁻ that may exist in basic conditions. Theinvention encompasses all such forms.

TLR agonists can exist as pharmaceutically acceptable salts. Thus, thecompounds may be used in the form of their pharmaceutically acceptablesalts i.e. physiologically or toxicologically tolerable salt (whichincludes, when appropriate, pharmaceutically acceptable base additionsalts and pharmaceutically acceptable acid addition salts).

In the case of TLR agonists shown herein which may exist in tautomericforms, the compound can be used in all such tautomeric forms.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encephalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE).

Meningococcal Protein Immunogens

NHBA (Neisserial Heparin Binding Antigen)

NHBA [181] was included in the published genome sequence formeningococcal serogroup B strain MC58 [26] as gene NMB2132 (GenBankaccession number GI:7227388; SEQ ID NO: 9 herein). Sequences of NHBAfrom many strains have been published since then. For example, allelicforms of NHBA (referred to as protein ‘287’) can be seen in FIGS. 5 and15 of reference 182, and in example 13 and FIG. 21 of reference 183 (SEQIDs 3179 to 3184 therein). Various immunogenic fragments of NHBA havealso been reported.

Preferred NHBA antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 9; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 9, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 9.

The most useful NHBA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 9. Advantageous NHBAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

One useful NHBA antigen comprises SEQ ID NO: 4, which is a fusion ofNHBA to NMB1030, as present in the BEXSERO™ product.

NadA (Neisserial Adhesin A)

The NadA antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [26] as gene NMB1994 (GenBankaccession number GI:7227256; SEQ ID NO: herein). The sequences of NadAantigen from many strains have been published since then, and theprotein's activity as a Neisserial adhesin has been well documented.Various immunogenic fragments of NadA have also been reported.

Preferred NadA antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 10; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 10, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 10.

NadA will usually be present in a composition in oligomeric form e.g.trimers [184].

The most useful NadA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 10. Advantageous NadAantigens for use with the invention can elicit bactericidalanti-mcningococcal antibodies after administration to a subject. SEQ IDNO: 6 is one such fragment, as present in the BEXSERO™ product.

fHbp (Factor H Binding Protein)

The fHbp antigen has been characterised in detail. It has also beenknown as protein ‘741’ [SEQ IDs 2535 & 2536 in ref. 183], ‘NMB1870’,‘GNA1870’ [185, 186, 207], ‘P2086’, ‘LP2086’ or ‘ORF2086’ [187-189]. Itis naturally a lipoprotein and is expressed across all meningococcalserogroups. The structure of fHbp's C-terminal immunodominant domain(‘fHbpC’) has been determined by NMR [190]. This part of the proteinforms an eight-stranded β-barrel, whose strands are connected by loopsof variable lengths. The barrel is preceded by a short α-helix and by aflexible N-terminal tail.

The fHbp antigen falls into three distinct variants [191] and it hasbeen found that serum raised against a given family is bactericidalwithin the same family, but is not active against strains which expressone of the other two families i.e. there is intra-familycross-protection, but not inter-family cross-protection. The inventioncan use a single fHbp variant, but is will usefully include a fHbp fromtwo or three of the variants.

Where a composition comprises a single fHBP variant, it may include oneof the following:

-   -   (a) a first polypeptide comprising a first amino acid sequence,        where the first amino acid sequence comprises an amino acid        sequence (i) having at least a % sequence identity to SEQ ID NO:        1 and/or (ii) consisting of a fragment of at least x contiguous        amino acids from SEQ ID NO: 1;    -   (b) a second polypeptide, comprising a second amino acid        sequence, where the second amino acid sequence comprises an        amino acid sequence (i) having at least b % sequence identity to        SEQ ID NO: 2 and/or (ii) consisting of a fragment of at least y        contiguous amino acids from SEQ ID NO: 2;    -   (c) a third polypeptide, comprising a third amino acid sequence,        where the third amino acid sequence comprises an amino acid        sequence (i) having at least c % sequence identity to SEQ ID NO:        3 and/or (ii) consisting of a fragment of at least z contiguous        amino acids from SEQ ID NO: 3.

The value of a is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96,97, 98, 99 or more. The value of h is at least 80 e.g. 82, 84, 86, 88,90, 92, 94, 95, 96, 97, 98, 99 or more. The value of c is at least 80e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The valuesof a, b and c may be the same or different. In some embodiments, a b andc are identical.

The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y isat least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 225, 250). The value of z is at least 7 e.g. 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 225, 250). The values of x, y and z may be the same or different.In some embodiments, x y and z are identical.

Fragments preferably comprise an epitope from the respective SEQ ID NO:sequence. Other useful fragments lack one or more amino acids (e.g. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminusand/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of the respective SEQ ID NO: whileretaining at least one epitope thereof.

In some embodiments the fragment of at least x contiguous amino acidsfrom SEQ ID NO: 1 is not also present within SEQ ID NO: 2 or within SEQID NO: 3. Similarly, the fragment of at least y contiguous amino acidsfrom SEQ ID NO: 2 might not also be present within SEQ ID NO: 1 orwithin SEQ ID NO: 3. Similarly, the fragment of at least z contiguousamino acids from SEQ ID NO: 3 might not also be present within SEQ IDNO: 1 or within SEQ ID NO: 2. In some embodiments, when said fragmentfrom one of SEQ ID NOs: 1 to 3 is aligned as a contiguous sequenceagainst the other two SEQ ID NOs, the identity between the fragment andeach of the other two SEQ ID NOs is less than 75% e.g. less than 70%,less than 65%, less than 60%, etc.

Where a composition comprises two different meningococcal fHBP antigens,it may include a combination of: (i) a first and second polypeptide asdefined above; (ii) a first and third polypeptide as defined above; or(iii) a second and third polypeptide as defined above. A combination ofa first and third polypeptide is preferred. Where a compositioncomprises two different meningococcal fHBP antigens, although these mayshare some sequences in common, the first, second and third polypeptideshave different fHBP amino acid sequences.

A polypeptide comprising the first amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type meningococcus protein which hasnascent amino acid sequence SEQ ID NO: 20 (MC58). In some embodimentssome or all of these antibodies do not bind to the wild-typemeningococcus protein which has nascent amino acid sequence SEQ ID NO:21 or to the wild-type meningococcus protein which has nascent aminoacid sequence SEQ ID NO: 22.

A polypeptide comprising the second amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type meningococcus protein which hasnascent amino acid sequence SEQ ID NO: 21 (2996). In some embodimentssome or all of these antibodies do not bind to the wild-typemeningococcus protein which has nascent amino acid sequence SEQ ID NO:20 or to the wild-type meningococcus protein which has nascent aminoacid sequence SEQ ID NO: 22.

A polypeptide comprising the third amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type meningococcus protein which hasnascent amino acid sequence SEQ ID NO: 22 (M1239). In some embodimentssome or all of these antibodies do not bind to the wild-typemeningococcus protein which has nascent amino acid sequence SEQ ID NO:20 or to the wild-type meningococcus protein which has nascent aminoacid sequence SEQ ID NO: 21.

A useful first amino acid sequence has at least 85% identity (e.g. >95%or 100%) to SEQ ID NO: 1 (strain MC58). Another useful first amino acidsequence has at least 95% identity (e.g. >98% or 100%) to SEQ ID NO: 23(strain CDC1573).

A useful third amino acid sequence has at least 85% identity (e.g. >95%or 100%) to SEQ ID NO: 3 (strain M1239). Another useful third amino acidsequence has at least 95% identity (e.g. >98% or 100%) to SEQ ID NO: 25(strain M98-250771).

Combinations comprising a mixture of first and third sequences basedaround SEQ ID NOs: 23 and 25 (or their close variants) are particularlyuseful. Thus a composition may comprise a polypeptide comprising aminoacid sequence SEQ ID NO: 24 and a polypeptide comprising amino acidsequence SEQ ID NO: 26.

Where a composition includes two meningococcal fHBP antigens, this maybe in a bivalent fl-IBP composition, or there may be more than twodifferent fl-IBP antigens e.g. in a trivalent or tetravalent fHBPcomposition.

Another useful fHbp which can be used according to the invention is oneof the modified forms disclosed, for example, in reference 192 e.g.comprising SEQ ID NO: 20 or 23 therefrom. These modified forms canelicit antibody responses which are broadly bactericidal againstmeningococci by recognising multiple fHbp variant. One such modifiedform is SEQ ID NO: 28 herein (SEQ ID NO: 23 in ref. 192), which can befused to non-fHbp sequences as disclosed in reference 193 e.g. to giveSEQ ID NO: 19 (which contains NMB2091 and two copies of SEQ ID NO: 28),which is used in the examples below.

SEQ ID NO: 77 from ref. 192 is another useful fHbp sequence which can beused in order to provide broad inter-strain reactivity.

In some embodiments fHBP polypeptide(s) are lipidated e.g. at aN-terminus cysteine. In other embodiments, however, fHBP polypeptide(s)are not lipidated. For lipidated fHBPs, lipids attached to cysteineswill usually include palmitoyl residues e.g. astripalmitoyl-S-glyceryl-cysteine (Pam3Cys), dipalmitoyl-S-glycerylcysteine (Pam2Cys), N-acetyl (dipalmitoyl-S-glyceryl cysteine), etc.Examples of mature lipidated f-BP sequences are SEQ ID NO: 24 (includingSEQ ID NO: 23) and SEQ ID NO: 26 (including SEQ ID NO: 25). If fHbpprotein(s) are located in a vesicle then they will usually be lipidated.

Administration of a fHBP will preferably elicit antibodies which canbind to a meningococcal polypeptide consisting of amino acid sequenceSEQ ID NO: 1, 2 or 3. Advantageous fHBP antigens for use with theinvention can elicit bactericidal anti-meningococcal antibodies afteradministration to a subject.

The total amount of a fHBP polypeptide will usually be between 1 and 500μg per unit dose e.g. between 60 and 200 μg per unit. An amount of 10,20, 40, 50, 60, 80, 100 or 200 μg per unit dose for each fHBPpolypeptide is typical in a human vaccine dose.

Where a composition comprises different meningococcal fHBP antigens,these may be present as separate polypeptides as described above (e.g. afirst and second polypeptide) or they may be present as part of a singlefusion polypeptide i.e. where at least two (e.g. 2, 3, 4, 5, or more)fHBP antigens are expressed as a single polypeptide chain, as disclosedfor meningococcal antigens in reference 194. Most usefully, a fusionpolypeptide can include each of a first, second and third sequence asdiscussed above e.g. SEQ ID NO: 27.

HmbR

The full-length HmbR sequence was included in the published genomesequence for meningococcal serogroup B strain MC58 [26] as gene NMB1668(SEQ ID NO: 7 herein). Reference 195 reports a HmbR sequence from adifferent strain (SEQ ID NO: 8 herein), and reference 196 reports afurther sequence (SEQ ID NO: 15 herein). SEQ ID NOs: 7 and 8 differ inlength by 1 amino acid and have 94.2% identity. SEQ ID NO: 15 is oneamino acid shorter than SEQ ID NO: 7 and they have 99% identity (oneinsertion, seven differences). The invention can use any such HmbRpolypeptide.

The invention can use a polypeptide that comprises a full-length HmbRsequence, but it will often use a polypeptide that comprises a partialHmbR sequence. Thus in some embodiments a HmbR sequence used accordingto the invention may comprise an amino acid sequence having at least i %sequence identity to SEQ ID NO: 7, where the value of i is 50, 60, 70,80, 90, 95, 99 or more. In other embodiments a HmbR sequence usedaccording to the invention may comprise a fragment of at least jconsecutive amino acids from SEQ ID NO: 7, where the value of j is 7, 8,10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more. In other embodiments a HmbR sequence used according tothe invention may comprise an amino acid sequence (i) having at least i% sequence identity to SEQ ID NO: 7 and/or (ii) comprising a fragment ofat least j consecutive amino acids from SEQ ID NO: 7.

Preferred fragments of j amino acids comprise an epitope from SEQ ID NO:7. Such epitopes will usually comprise amino acids that are located onthe surface of HmbR. Useful epitopes include those with amino acidsinvolved in HmbR's binding to haemoglobin, as antibodies that bind tothese epitopes can block the ability of a bacterium to bind to hosthaemoglobin. The topology of HmbR, and its critical functional residues,were investigated in reference 197. Fragments that retain atransmembrane sequence are useful, because they can be displayed on thebacterial surface e.g. in vesicles. If soluble HmbR is used, however,sequences omitting the transmembrane sequence, but typically retainingepitope(s) from the extracellular portion, can be used.

The most useful HmbR antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 7. Advantageous HmbRantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

NspA (Neisserial Surface Protein A)

The NspA antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [26] as gene NMB0663 (GenBankaccession number GI:7225888; SEQ ID NO: 11 herein). The antigen waspreviously known from references 198 & 199. The sequences of NspAantigen from many strains have been published since then. Variousimmunogenic fragments of NspA have also been reported.

Preferred NspA antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 11; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 11, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 11.

The most useful NspA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 11. Advantageous NspAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

NhhA (Neisseria Hia Homologue)

The NhhA antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [26] as gene NMB0992 (GenBankaccession number GI:7226232; SEQ ID NO: 12 herein). The sequences ofNhhA antigen from many strains have been published since e.g. refs 182 &200, and various immunogenic fragments of NhhA have been reported. It isalso known as Hsf.

Preferred NhhA antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 12; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 12, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 12.

The most useful NhhA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 12.

Advantageous NhhA antigens for use with the invention can elicitbactericidal anti-meningococcal antibodies after administration to asubject.

App (Adhesion and Penetration Protein)

The App antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [26] as gene NMB1985 (GenBankaccession number GI:7227246; SEQ ID NO: 13 herein). The sequences of Appantigen from many strains have been published since then. It has alsobeen known as ‘ORF1’ and ‘Hap’. Various immunogenic fragments of Apphave also been reported.

Preferred App antigens for use with the invention comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 13; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 13, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 13.

The most useful App antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 13. Advantageous Appantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

Omp85 (85 kDa Outer Membrane Protein)

The Omp85 antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [26] as gene NMB0182 (GenBankaccession number GI:7225401; SEQ ID NO: 14 herein). The sequences ofOmp85 antigen from many strains have been published since then. Furtherinformation on Omp85 can be found in references 201 and 202. Variousimmunogenic fragments of Omp85 have also been reported.

Preferred Omp85 antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 14; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 14, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 14.

The most useful Omp85 antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 14. Advantageous Omp85antigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

TbpA

The TbpA antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [26] as gene NMB0461 (GenBankaccession number GI:7225687; SEQ ID NO: 23 herein). The sequences ofTbpA from many strains have been published since then. Variousimmunogenic fragments of TbpA have also been reported.

Preferred TbpA antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 23; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 23, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 23.

The most useful TbpA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 23. Advantageous TbpAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

TbpB

The TbpB antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [26] as gene NMB0460 (GenBankaccession number GI:7225686; SEQ ID NO: 24 herein). The sequences ofTbpB from many strains have been published since then. Variousimmunogenic fragments of TbpB have also been reported.

Preferred TbpB antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 24; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 24, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 24.

The most useful TbpB antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 24. Advantageous TbpBantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

Cu,Zn-Superoxide Dismutase

The Cu,Zn-superoxide dismutase antigen was included in the publishedgenome sequence for meningococcal serogroup B strain MC58 [26] as geneNMB1398 (GenBank accession number GI:7226637; SEQ ID NO: 25 herein). Thesequences of Cu,Zn-superoxide dismutase from many strains have beenpublished since then. Various immunogenic fragments of Cu,Zn-superoxidedismutase have also been reported.

Preferred Cu,Zn-superoxide dismutase antigens for use with the inventioncomprise an amino acid sequence: (a) having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5% or more) to SEQ ID NO: 25; and/or (b) comprising afragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 25,wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragmentsof (b) comprise an epitope from SEQ ID NO: 25.

The most useful Cu,Zn-superoxide dismutase antigens can elicitantibodies which, after administration to a subject, can bind to ameningococcal polypeptide consisting of amino acid sequence SEQ ID NO:25. Advantageous Cu,Zn-superoxide dismutase antigens for use with theinvention can elicit bactericidal anti-meningococcal antibodies afteradministration to a subject.

ZnuD

The ZnuD antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [26] as gene NMB0964 (GenBankaccession number GI:15676857; SEQ ID NO: 29 herein). The sequences ofZnuD from many strains have been published since then e.g. seereferences 203 & 204.

Preferred ZnuD antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 29; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 29, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 29.

The most useful ZnuD antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 29.

Advantageous ZnuD antigens for use with the invention can elicitbactericidal anti-meningococcal antibodies after administration to asubject.

Meningococcal Vesicles

The invention can be used with various types of vesicle which are knownfor Neisseria meningitidis.

Reference 22 discloses the construction of vesicles from meningococcalstrains modified to express six different PorA subtypes. References205-207 report pre-clinical studies of an OMV vaccine in which fHbp(also known as GN1870) is over-expressed (and this over-expression canbe combined with knockout of LpxL1 [208]). Reference 209 recentlyreported a clinical study of five formulations of an OMV vaccine inwhich PorA & FrpB are knocked-out and Hsf & TbpA are over-expressed.Reference 210 reports a native outer membrane vesicle vaccine preparedfrom bacteria having inactivated synX, lpxL1, and lgtA genes. All suchvesicles can be used herein.

OMVs can be prepared from meningococci which over-express desiredantigen(s) due to genetic modification. In addition to geneticmodification(s) which cause over-expression of antigen(s) of interest,the bacteria may include one or more further modifications. Forinstance, the bacterium may have a knockout of one or more of lpxL1,lgtB, porA, frpB, synX, lgtA, mlt4 and/or lst.

The bacterium may have low endotoxin levels, achieved by knockout ofenzymes involved in LPS biosynthesis [211,212].

The bacterium may be of any serotype (e.g. 1, 2a, 2b, 4, 14, 15, 16,etc.), any serosubtype, and any immunotype (e.g. L1; L2; L3; L3,3,7;L10; etc.). Vesicles can usefully be prepared from strains having one ofthe following subtypes: P1.2; P1.2,5; P1.4; P1.5; P1.5,2; P1.5,c;P1.5c,10; P1.7,16; P1.7,16b; P1.7h,4; P1.9; P1.15; P1.9,15; P1.12,13;P1.13; P1.14; P1.21,16; P1.22,14.

The bacterium may be from any suitable lineage, including hyperinvasiveand hypervirulent lineages e.g. any of the following seven hypervirulentlineages: subgroup I; subgroup III; subgroup IV-1; ET-5 complex; ET-37complex; A4 cluster; lineage 3. These lineages have been defined bymultilocus enzyme electrophoresis (MLEE), but multilocus sequence typing(MLST) has also been used to classify meningococci [ref. 213] e.g. theET-37 complex is the ST-II complex by MLST, the ET-5 complex is ST-32(ET-5), lineage 3 is ST-41/44, etc.

In some embodiments a bacterium may include one or more of the knockoutand/or hyper-expression mutations disclosed in references 226 and214-216. Suitable genes for modification include: (a) Cps, CtrA, CtrB,CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PilC,PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB [214]; (b) CtrA, CtrB,CtrC, CtrD, FrpB, GalE, IHtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PhoP,PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB; (c) ExbB,ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, PilC, PorB,SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB; and (d) CtrA, CtrB, CtrD,FrpB, OpA, OpC, PilC, PorB, SiaD, SynA, SynB, and/or SynC.

A bacterium may have one or more, or all, of the followingcharacteristics: (i) down-regulated or knocked-out LgtB and/or GalE totruncate the meningococcal LOS; (ii) up-regulated TbpA; (iii)up-regulated NhhA; (iv) up-regulated Omp85; (v) up-regulated LbpA; (vi)up-regulated NspA; (vii) knocked-out PorA; (viii) down-regulated orknocked-out FrpB; (ix) down-regulated or knocked-out Opa; (x)down-regulated or knocked-out Opc; (xi) deleted cps gene complex; (xi)up-regulated NHBA; (xii) up-regulated NadA; (xiii) up-regulated NHBA andNadA; (xiv) up-regulated fHbp; (xv) down-regulated LpxL1. A truncatedLOS can be one that does not include a sialyl-lacto-N-neotetraoseepitope e.g. it might be a galactose-deficient LOS. The LOS may have noa chain.

If lipo-oligosaccharide (LOS) is present in a vesicle it is possible totreat the vesicle so as to link its LOS and protein components(“intra-bleb” conjugation [216]).

The vesicles may lack LOS altogether, or they may lack hexa-acylated LOSe.g. LOS in the vesicles may have a reduced number of secondary acylchains per LOS molecule [217]. For example, the vesicles may from astrain which has a lpxL1 deletion or mutation which results inproduction of a penta-acylated LOS [206,210]. LOS in a strain may lack alacto-N-ncotetraose epitope e.g. it may be a lst and/or lgtB knockoutstrain [209]. LOS may lack at least one wild-type primary O-linked fattyacid [218]. LOS having. The LOS may have no a chain. The LOS maycomprise GlcNAc-Hep₂phosphoethanolamine-KDO₂-Lipid A [219].

As a result of up-regulation mentioned above, vesicles prepared frommodified meningococci contain higher levels of the up-regulatedantigen(s). The increase in expression in the vesicles (measuredrelative to a corresponding wild-type strain) is usefully at least 10%,measured in mass of the relevant antigen per unit mass of vesicle, andis more usefully at least 20%, 30%, 40%, 50%, 75%, 100% or more.

Suitable recombinant modifications which can be used to causeup-regulation of an antigen include, but are not limited to: (i)promoter replacement; (ii) gene addition; (iii) gene replacement; or(iv) repressor knockout. In promoter replacement, the promoter whichcontrols expression of the antigen's gene in a bacterium is replacedwith a promoter which provides higher levels of expression. Forinstance, the gene might be placed under the control of a promoter froma housekeeping metabolic gene. In other embodiments, the antigen's geneis placed under the control of a constitutive or inducible promoter.Similarly, the gene can be modified to ensure that its expression is notsubject to phase variation. Methods for reducing or eliminating phasevariability of gene expression in meningococcus are disclosed inreference 220. These methods include promoter replacement, or theremoval or replacement of a DNA motif which is responsible for a gene'sphase variability. In gene addition, a bacterium which already expressesthe antigen receives a second copy of the relevant gene. This secondcopy can be integrated into the bacterial chromosome or can be on anepisomal element such as a plasmid. The second copy can have a strongerpromoter than the existing copy. The gene can be placed under thecontrol of a constitutive or inducible promoter. The effect of the geneaddition is to increase the amount of expressed antigen. In genereplacement, gene addition occurs but is accompanied by deletion of theexisting copy of the gene. For instance, this approach was used inreference 207, where a bacterium's endogenous chromosomal fHbp gene wasdeleted and replaced by a plasmid-encoded copy (see also reference 221).Expression from the replacement copy is higher than from the previouscopy, thus leading to up-regulation. In repressor knockout, a proteinwhich represses expression of an antigen of interest is knocked out.Thus the repression does not occur and the antigen of interest can beexpressed at a higher level. Promoters for up-regulated genes canadvantageously include a CREN [222].

A modified strain will generally be isogenic with its parent strain,except for a genetic modification. As a result of the modification,expression of the antigen of interest in the modified strain is higher(under the same conditions) than in the parent strain. A typicalmodification will be to place a gene under the control of a promoterwith which it is not found in nature and/or to knockout a gene whichencodes a repressor.

In embodiments where NHBA is up-regulated, various approaches can beused. For convenience, the approach already reported in reference 181can be used i.e. introduction of a NHBA gene under the control of anIPTG-inducible promoter. By this approach the level of expression ofNHBA can be proportional to the concentration of IPTG added to aculture. The promoter may include a CREN.

In embodiments where NadA is up-regulated, various approaches can beused. One useful approach involves deletion of the gene encoding NadR(NMB1843), which is a transcriptional repressor protein [223] whichdown-regulates or represses the NadA-encoding gene in all strainstested. Knockout of NadR results in high-level constitutive expressionof NadA. An alternative approach to achieve NadA up-regulation is to add4-hydroxyphenylacetic to the culture medium. A further approach is tointroduce a NadA gene under the control of an IPTG-inducible promoter.

Up-regulation of NhhA is already reported in references 209 and 224.Up-regulation of TbpA is already reported in references 209, 224 and225. Up-regulation of HmbR is already reported in reference 196.Up-regulation of TbpB is already reported in reference 225.Up-regulation of NspA is already reported in reference 226, incombination with porA4 and cps knockout. Up-regulation ofCu,Zn-superoxide dismutase is already reported in reference 225.Up-regulation of fHbp is already reported in references 205-207 & 221,and by a different approach (expressing a constitutively-active mutantFNR) in references 227 & 228.

In some embodiments each of NHBA, NadA and fHbp are up-regulated. Thesethree antigens are components of the “universal vaccine” disclosed inreference 8 or “4CMenB” [229,230]. In one embodiment, expression of NHBAis controlled by a strong promoter, NadR is knocked out, and the strainexpresses a constitutively active mutant FNR. In another embodiment,expression of NHBA is controlled by a strong promoter, expression offHbp is controlled by a strong promoter, and NadR is knocked out. Thebacterium can also be a bacterium which does not express an active MltA(GNA33), such that it spontaneously releases vesicles which containNHBA, NadA and fHbp. Ideally, the bacterium does not express a nativeLPS e.g. it has a mutant or knockout of LpxL1.

The vesicles may include one, more than one, or (preferably) zero PorAserosubtypes. Modification of meningococcus to provide multi-PorA OMVsis known e.g. from references 22 and 23. Conversely, modification toremove PorA is also known e.g. from reference 209.

The vesicles may be free from one of both of PorA and FrpB. Preferredvesicles are PorA-free.

The invention may be used with mixtures of vesicles from differentstrains. For instance, reference 24 discloses vaccine comprisingmultivalent meningococcal vesicle compositions, comprising a firstvesicle derived from a meningococcal strain with a serosubtype prevalentin a country of use, and a second vesicle derived from a strain thatneed not have a serosubtype prevent in a country of use. Reference 25also discloses useful combinations of different vesicles. A combinationof vesicles from strains in each of the L2 and L3 immunotypes may beused in some embodiments.

Another useful combination of vesicles is disclosed in references 231 &232. A trivalent mixture of this type can include vesicles prepared fromeach of: (a) a first strain which over-expresses NadA; (b) a secondstrain which over-expresses a fHbp sequence from variant 1 i.e. a firstfHbp polypeptide sequence as defined above; and (c) a third strain whichover-expresses a fHbp sequence from variant 2 i.e. a second fHbppolypeptide sequence as defined above. These strains can also have othermodifications e.g. knockout of synX and LpxLJ, as disclosed in ref. 231.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-B show serum total IgG responses against tetanus toxoid. FIG.1A shows the serum total IgG responses against tetanus toxoid at day 35.FIG. 1B shows the serum total IgG responses against tetanus toxoid atday 49.

FIGS. 2A-B show serum total IgG responses against diphtheria toxoid.FIG. 2A shows the serum total IgG responses against diphtheria toxoid atday 35. FIG. 2B shows the serum total IgG responses against diphtheriatoxoid at day 49.

FIGS. 3A-B show serum total IgG responses against pertussis toxoid. FIG.3A shows the serum total IgG responses against pertussis toxoid at day35. FIG. 3B shows the serum total IgG responses against pertussis toxoidat day 49.

FIGS. 4A-B show serum total IgG responses against pertactin. FIG. 4Ashows the serum total IgG responses against pertactin at day 35. FIG. 4Bshows the serum total IgG responses against pertactin at day 49.

FIGS. 5A-B show serum total IgG responses against FHA. FIG. 5A shows theserum total IgG responses against FHA at day 35. FIG. 5B shows the serumtotal IgG responses against FHA at day 49.

FIG. 6 shows serum total IgG responses against NadA.

FIG. 7 shows serum total IgG responses against NHBA.

FIG. 8 shows serum total IgG responses against fHbp.

The y-axis scale in all cases is 0.01 to 10,000.

MODES FOR CARRYING OUT THE INVENTION

An immunogen combination was prepared, containing the followingcomponents:

Immunogen Amount (per 0.5 ml) T Tetanus toxoid 5 Lf D Diphtheria toxoid2 Lf aP Pertussis toxoid, PT-9K/129G 4 μg FHA 4 μg Pertactin 8 μg MenBNHBA (SEQ ID NO: 4) 50 μg NadA (SEQ ID NO: 6) 50 μg fHbp (SEQ ID NO: 19)50 μg

For comparison purposes, an equivalent combination was prepared butwithout the MenB proteins.

These two immunogen combinations are referred to as “TdaP-MenB” and“TdaP”.

These two combinations were adjuvanted with:

-   -   (a) aluminium hydroxide, 1 mg/dose (“AI-H”)    -   (b) aluminium hydroxide, 1 mg/dose, with 100 μg adsorbed ‘K2’        TLR7 agonist    -   (c) aluminium hydroxide, 1 mg/dose, with 100 μg adsorbed        synthetic MPL TLR4 agonist (d) MF59 squalene-containing        oil-in-water emulsion.

All antigens were adsorbed to the Al—H in compositions (a) to (c) forboth TdaP and TdaP-MenB, although pertactin was not fully adsorbed incompositions which include the MenB immunogens.

In addition to these four pairs of adjuvanted compositions, a furtherpair was unadjuvanted. This gave 10 compositions in total, (C1) to(C10):

No adjuvant Al—H Al—H/K2 Al—H/MPL MF59 TdaP C1 C2 C3 C4 C5 TdaP-MenB C6C7 C8 C9 C10

Furthermore, for comparison the BOOSTRIX™ product was also tested (“C”),which contains (per 0.5 ml) 2.5Lf of diphtheria toxoid, 5Lf tetanustoxoid, and 18.5 μg acellular pertussis antigens (a mixture of purifiedPT, FHA and p69 pertactin), adjuvanted with a mixture of aluminiumphosphate and hydroxide salts. Finally, an immunogen-free negativecontrol of buffer alone was also prepared (“C12”).

These 12 compositions were administered to female Balb/C mice (6 weeksold) at 100 μl intramuscular doses (2×50 μl) on days 0, 21 and 35. Serawere tested 2 weeks after each dose and assessed for specific IgGresponses against each of the 8 immunogens (except that only C6-C10 &C12 were tested for responses against the 3 MenB immunogens). Thesetiters are shown in FIGS. 1-8. FIGS. 1-5 show data for days 35 (1A to5A) and 49 (1B to 5B), whereas FIGS. 6-8 show data only for day 35.

The data show that the MenB antigens have no negative impact on IgGresponses against the diphtheria, tetanus and acellular pertussisantigens after 2 or 3 doses. Furthermore, the inclusion of a TLR agonistwith the Al—H adjuvant improved IgG responses against all antigens. Theemulsion adjuvant also gave better results than Al—H alone. In all caseshowever, the adjuvants did not have a large impact on anti-PT responses.

The second dose of vaccine (day 21) led to an increase of IgG responseagainst all antigens, but the third dose (day 35) did not provide afurther significant increase. Thus the studied adjuvants provide a morerapid response to the re-injected antigens, which can be very useful inbooster situations.

Thus the mixture of D, T, aP and MenB antigens offers a new andeffective combination vaccine.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

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We claim: 1: An immunogenic composition, comprising: (a) a serogroup Bmeningococcus immunogen; and (b) a diphtheria toxoid, a tetanus toxoid,and a pertussis toxoid, wherein the diphtheria toxoid is present in anexcess relative to tetanus toxoid as measured in Lf units. 2: Thecomposition of claim 1, further comprising an adjuvant.